疫苗製備方法和抗腫瘤疫苗的製作方法
2023-05-04 09:45:21
專利名稱：疫苗製備方法和抗腫瘤疫苗的製作方法
技術領域：
本發明涉及生物技術領域，具體而言涉及一種疫苗製備方法和製備的生物疫苗，特別是新型的抗腫瘤疫苗，如EGFR分子疫苗。分子疫苗屬新生物技術藥物(New Biotechnological drug)範疇，涉及到多種生物技術，如PCR技術、分子克隆技術、基因表達技術、現代疫苗技術、以及生物技術藥物學和藥劑學等。
背景技術：
疫苗(vaccine)是一種能刺激機體免疫系統產生抗特異性靶物質(如病毒、細菌等)的免疫反應的物質。經典的疫苗概念來源於抗感染免疫，它主要是使用處理過的病原微生物(如病毒、細菌等)及其衍生物免疫機體產生體液免疫反應以預防感染性疾病。比如，滅活疫苗(inactivated vaccine)是利用化學方法殺死實際感染成分的衍生物。弱毒疫苗(attenuated vaccine)是改變過的活病毒或細菌使其在接種的生物體內不能增殖。這兩類疫苗都是通過表面蛋白(抗原)作用於B和T淋病細胞來進行免疫的。當病原體生物感染人體時，B和T淋病細胞就會迅速作出反應，在病原微生物產生破壞之前將其消滅。然而，這兩類疫苗潛藏著危險，因為它們能被傳染性病菌所汙染。例如，每年總有一小部分兒童因為接種脊髓灰質炎疫苗而感染此病。因此，一個發展方向是亞單位疫苗(subunit vaccine)。亞單位疫苗是利用DNA重組技術生產的單純由表面蛋白組成、能引起免疫系統反應的疫苗，也稱為基因工程疫苗或者DNA重組疫苗。參見，基因工程學原理，馬建崗，主編，西安交通大學出版社，p240，2001.11。除了上述採用病原體本身、其具有免疫原性的構成成分、以及基因表達產物外，也有人研究使用「裸」DNA作為疫苗的基因疫苗，是由來源於病原體的一個抗原編碼基因及作為其載體的質粒DNA組成。通過注射或粒子轟擊等途徑將基因疫苗導入人體後，這段基因可以在活體細胞中合成抗原蛋白，從而引起機體免疫反應。目前，基因表達文庫免疫技術是發現免疫活性基因的最系統和客觀的手段。參見，第四次浪潮生物經濟，封展旗，楊同衛，編著，經濟管理出版社，p169-171，2000。
疫苗的主要作用在於預防疾病。現在也有應用疫苗來治療疾病。所以，針對正常的人體和動物體而言，疫苗的作用在於增強機體的防病和抗病能力，起到預防疾病的目的；對於患有某種疾病或者病患的人體和動物體而言，疫苗的作用在於誘導機體產生針對特定致病因素的反應，達到消除病灶，治療疾病或者病患的目的。可以認為疫苗就是通過影響機體的免疫系統達到預防，或者治療，或者預防和治療疾病或者病患的物質的總稱。
動物體內細胞分裂調節失控而無限增殖的細胞稱為腫瘤細胞(tumor cell)。具有轉移能力的腫瘤稱為惡性腫瘤(malignancy)，上皮組織的惡性腫瘤稱為癌。參見，細胞生物學，翟中和等，主編，高等教育出版社，p423-427，2000(2001重印)。對於腫瘤的起源和宿主對於腫瘤的應答已經進行了大量的基礎研究和臨床應用研究。關於起源，已經顯示大量的環境因素對於動物具有致癌性和致突變性。有些腫瘤事實上與暴露於某種特定的物質有關(例如石棉與造船廠工人的間皮瘤)。已知病毒也可以誘發動物腫瘤，例如B型肝炎病毒與肝癌有關。宿主免疫應答在許多情況下產生以抵抗腫瘤，而在某些情況下可能是保護性的。
腫瘤細胞和正常細胞可以根據其抗原的質和量的差異區分開來。腫瘤細胞的抗原，特別是腫瘤特異抗原為腫瘤細胞特有，成為腫瘤預防和治療中的研究重點。
用於腫瘤治療的腫瘤疫苗(cancer vaccine)尤其具有重要的意義。這種治療性疫苗不同於傳統的預防性疫苗，主要用於治療已罹患癌症的病人，其目的以激發患者機體對腫瘤的特異性免疫應答，最終達到有效的排斥。研製和開發新型腫瘤疫苗已成為近年國際上腫瘤免疫治療的熱點，主要包括腫瘤細胞疫苗、基因修飾疫苗、多肽瘤苗和基因/DNA疫苗等。
腫瘤細胞疫苗是將病人或者患病動物的腫瘤細胞通過物理或者化學方法處理後的具有治療或者輔助治療作用的完整的死細胞。處理方法可以是X光照射，有機溶劑處理等。這種疫苗被通過注射等方法施用於機體後，可以誘導或者增強病人對其腫瘤的免疫力。基因修飾疫苗、多肽瘤苗和基因/DNA疫苗也均是通過利用腫瘤抗原及其片段，或者編碼腫瘤抗原及其片段的多核苷酸，以及含有這些多核苷酸的載體或者細胞對於腫瘤進行治療或者輔助治療的疫苗。
如上所述，疫苗在疾病預防和/或治療中具有重要作用。腫瘤作為一種嚴重的動物體細胞異常生長現象，隨著環境汙染的加劇，發生頻率有所增強。因此開發新的疫苗，特別是腫瘤疫苗是必須的。本發明提供了一種製備疫苗的新方法，提供了一種新型腫瘤疫苗-EGFR分子疫苗(EGFR molecular vaccine)，滿足了上述需要。
發明概述第一個方面，本發明提供了一種製備疫苗的方法。該方法包括1)分析特定病原體的特異抗原；2)獲得編碼特異抗原的多核苷酸序列；3)獲得與該多核苷酸序列有足夠差異的多核苷酸序列；4)利用步驟3)中所得的多核苷酸序列製備疫苗。
第二個發麵，本發明提供了一種製備腫瘤疫苗的方法。該方法包括
1)分析動物體內的腫瘤細胞的特異抗原；2)獲得編碼特異抗原的多核苷酸序列；3)獲得與該多核苷酸序列有足夠差異的多核苷酸序列；4)利用步驟3)中所得的多核苷酸序列製備疫苗。
第三個方面，本發明提供了一種製備EGFR疫苗的方法。該方法包括1)分析動物體內的腫瘤細胞的EGFR抗原；2)獲得編碼特異抗原的多核苷酸序列；3)獲得與該多核苷酸序列有足夠差異的多核苷酸序列；4)利用步驟3)中所得的多核苷酸序列製備疫苗。
第四個方面，本發明提供了一種核酸疫苗，其中含有與編碼一種病原體上的一種特定抗原的多核苷酸序列有一定的差異，而且其同源性需要達到一定數值的多核苷酸序列。疫苗中所含多核苷酸序列與病原體中該特定抗原的多核苷酸序列所編碼的多肽的同源性為30-95％。
第五個方面，本發明提供了一種核酸疫苗，其中含有與編碼一種病原體上的兩種或者兩種以上特定抗原的多核苷酸序列有一定的差異，而且其同源性分別要達到一定數值的兩種或者兩種以上多核苷酸序列。疫苗中所含多核苷酸序列與病原體中相應的特定抗原的多核苷酸序列所編碼的多肽的同源性為30-95％。
第六個方面，本發明提供了一種蛋白質或者多肽疫苗，其中含有與一種病原體上的一種特定抗原的蛋白質部分中的胺基酸序列有一定的差異，而且其同源性為30-95％的蛋白質或者多肽分子。
第七個方面，本發明提供了一種蛋白質或者多肽疫苗，其中含有與一種病原體上的兩種或者兩種以上特定抗原的蛋白質部分中的胺基酸序列有一定的差異，而且其同源性為30-95％的兩種或者兩種以上蛋白質或者多肽分子。
第八個方面，本發明提供了一種表皮生長因子受體(EGFR)核酸疫苗，其中含有與編碼一種生物體內的EGFR分子的基因中的核苷酸序列有一定的差異，而且其同源性需要達到一定數值的一種多核苷酸序列。疫苗中所含多核苷酸序列編碼的表皮生長因子受體與該生物體內的表皮生長因子受體中的胺基酸序列的同源性為30-95％。
第九個方面，本發明提供了一種表皮生長因子受體(EGFR)核酸疫苗，其中含有與編碼一種生物體內的EGFR分子的基因中的核苷酸序列有一定的差異，而且其同源性需要達到一定數值的兩種或者兩種以上的多核苷酸序列。疫苗中所含多核苷酸序列編碼的表皮生長因子受體與該生物體內的表皮生長因子受體中的胺基酸序列的同源性為30-95％。疫苗中所含的兩種或者兩種以上的多核苷酸序列可以具有不同的來源，其與該生物體內的EGFR分子的基因中的核苷酸序列的同源性可以不同。
第十個方面，本發明提供了一種表皮生長因子受體(EGFR)蛋白質或者多肽疫苗，其中含有與一種生物體內的EGFR分子的變體。疫苗中所含表皮生長因子受體的胺基酸序列與該生物體內的表皮生長因子受體中的胺基酸序列的同源性為30-95％。
第十個方面，本發明提供了一種表皮生長因子受體(EGFR)蛋白質或者多肽疫苗，其中含有與兩種或者兩種以上的與生物體內EGFR分子不同的變體。疫苗中所含表皮生長因子受體的胺基酸序列與該生物體內的表皮生長因子受體中的胺基酸序列的同源性為30-95％。
第十一個方面，本發明提供了的表皮生長因子受體核酸疫苗和表皮生長因子受體蛋白質或者多肽疫苗用於腫瘤預防和治療中的用途。
發明祥述在此所用，「病原體」是指侵入生物體的外來生物體，包括細菌、病毒、真菌、原蟲、蠕蟲等，它們可以侵入另外的生物體中，導致一定的疾病或者疾病狀態。
在此所用，「腫瘤細胞」是指生物體內發生了生長和分裂失控，脫離了衰老和死亡的正常途徑的細胞。「腫瘤細胞」群構成腫瘤組織。在本發明中，術語「腫瘤」是以其廣義而言，包括任何類型的細胞不正常的增殖，也指腫瘤組織或者腫瘤細胞，需要對這種增殖予以遏制或者將這些細胞以及組織予以抑制或者消除。
在此所用，「生物體」是指動物體，尤其指「人體」。對於存在有免疫系統的動物，本發明的方法和疫苗均可以適用。
可以利用本技術領域的基礎研究結果或者已知的方法確定病原體或者腫瘤細胞中的特定抗原作為製備本發明所述疫苗的靶標。由於特定病原體或者體內的異常細胞具有特定的標記分子，比如它們的特定抗原，所以，以這些分子作為靶標，可以設計疫苗來激發生物體針對這些有害物質、生物或者細胞的免疫反應，達到防病和治病的目的。
從GenBank等公開的資料庫，或者眾多的網站上可以找到相應的有用抗原的基因序列，或者其胺基酸序列。根據中心法則，利用胺基酸序列或者基因序列進行推導所得的序列也可以應用於本發明中。有用的網站包括，但不限於，http//www.sanger.ac.uk/Proiects/Microbes.http//bioweb.pasteur.fr/Genoist/TubercuList.
在本發明中，使用的針對靶抗原的核酸序列或者多肽的胺基酸序列不同於編碼該靶抗原的蛋白質或者多肽部分的核酸序列或者其胺基酸序列。其同源性以編碼產物而言為30-95％。
同源性通常用序列分析軟體來測定。在上下文中對於兩種多肽序列，術語「同源性」指相同或具有特定百分比的胺基酸殘基的兩個或多個序列，當在同一比較窗口或指定區域內儘可能一致地比較或對齊時，所述胺基酸殘基是相同的，這正如用大量序列比較算法或通過人工對齊和視覺觀察所測定那樣。
對於序列比較，通常一個序列作為參考序列，測試序列與其進行比較。當使用序列比較算法時，將測試序列和參考序列輸入計算機，指定序列算法程序參數。然後用序列比較算法基於程序參數計算測試序列相對於參考序列的序列同源性百分比。在本發明中，某種選定的抗原中的胺基酸序列作為參考序列，疫苗中核苷酸序列的編碼序列或者疫苗中的蛋白質或者多肽中的胺基酸序列就是測試序列，該測試序列與參考序列的同源性是本發明的一個方面。
如果將靶抗原的核酸序列或者多肽的胺基酸序列看作「母序列或者參考序列」，那麼本發明所用的核酸序列或者多肽的胺基酸序列可以看作「子序列或者衍生序列」。
「母序列或者參考序列」的來源可以是現有技術，比如上面提到的資料庫，網站，公開的發明和論文等。它們也可以來源於對特定抗原，不論微生物抗原還是體內腫瘤的抗原的研究。可以是對基因組，染色體，蛋白質或者多肽，mRNA，DNA分子進行研究和分析獲得母序列或者參考序列或者其序列信息。
「子序列或者衍生序列」是由「母序列或者參考序列」演變而來的序列，它們與「母序列或者參考序列」有一定的差異，但是又不是完全不同。「子序列或者衍生序列」可以是自然界已經存在的與「母序列或者參考序列」具有結構上關聯的序列，也可以是通過人工方法得到的序列。前者是指不同生物或者生物個體內存在的編碼同源蛋白的序列。比如，人表皮生長因子受體的編碼序列和小鼠表皮生長因子受體的編碼序列，可以將它們看作本發明所稱「母序列」與「子序列」的關係，或者「參考序列」與「衍生序列」的關係。這種稱呼可以相互交換。比如將小鼠表皮生長因子受體的編碼序列看作「母序列」，則人表皮生長因子受體的編碼序列被看作「子序列」；或者人表皮生長因子受體的編碼序列被看作「母序列」，小鼠表皮生長因子受體的編碼序列看作「子序列」。也可以以「母序列或者參考序列」為起始物質，經過突變或者修飾得到「子序列或者衍生序列」。還可以通過人工合成方法得到與「母序列或者參考序列」有點差異的「子序列或者衍生序列」。通過檢索有關資料庫，在計算機上運行有關程序得到「母序列或者參考序列」的「子序列或者衍生序列」，然後通過人工合成得到具有「子序列或者衍生序列」的核酸、蛋白質以及其片段的物質。通過篩選有關分子文庫，也可以得到本發明所稱「子序列或者衍生序列」。具有「子序列或者衍生序列」的核酸，核酸片段，寡核苷酸，多核苷酸以及具有「子序列或者衍生序列」的蛋白質，多肽，及其片段可以用於本發明的實施中。在本發明實施中，對這些具有「子序列或者衍生序列」的核酸，核酸片段，寡核苷酸，多核苷酸以及具有「子序列或者衍生序列」的蛋白質，多肽，及其片段可以進行各種修飾。也可以將它們包括在適當的載體或者細胞或者病毒中，達到本發明的發明目的。上述用於本發明實施中的各種物質可以是分離的，可以是純化的；它們之間也可以是混合的，還可以混合進一定物質，比如佐劑。
此處所用，短語「核酸」或「核酸序列」指寡核苷酸、核苷酸、多核苷酸，或指寡核苷酸、核苷酸、多核苷酸中任一種的片段，指基因組或合成來源的DNA或RNA，指肽核酸(PNA)，或者指任何天然或合成來源的類似DNA或類似RNA的物質。
特定多肽或蛋白質的「編碼序列」或「編碼特定多肽或蛋白質的核苷酸序列」是當在置於適當調節序列控制下時被轉錄或翻譯到多肽或蛋白質中的核酸序列。
術語「基因」指涉及產生多肽鏈的DNA片段；包括編碼區之前和之後的區(前導區和非轉錄尾區)，以及適當的時候，包括個體編碼片段(外顯子)之間的間插序列(內含子)。
正如此處所用，「胺基酸」或「胺基酸序列」指寡肽、肽、多肽或蛋白序列，或指寡肽、肽、多肽或蛋白序列中任意一種的片段、部分或亞單位，指天然分子或合成分子。
正如此處所用，術語「多肽」指通過肽鍵或修飾的肽鍵即肽等排物(peptideisostere)彼此連接在一起的胺基酸，並且可以含有除了20個由基因編碼的胺基酸以外的修飾胺基酸。多肽可以通過任一種天然方法修飾，如翻譯後加工，或通過本技術領域已知的化學修飾技術修飾。修飾可以發生在多肽中的任何地方，包括肽骨架、胺基酸側鏈和氨基末端或羧基末端。應該意識到，在給定的多肽中，相同類型的修飾可以在數個位點以相同或變化的程度存在。而且，給定的多肽可以具有許多類型的修飾。這些修飾包括乙醯化作用、醯化作用、ADP-核糖基化作用、醯胺化作用、黃素的共價附著、亞鐵血紅素部分的共價附著、核苷酸或核苷酸衍生物的共價附著、脂質或脂質衍生物的共價附著、交聯環化、二硫鍵形成、脫甲基作用、共價交聯的形成、半胱氨酸的形成、焦穀氨酸的形成、甲醯化作用、γ-羧化作用、糖基化作用、GPI錨形成、羥基化作用、碘化作用、甲基化作用、豆蔻醯化、氧化、蛋白水解加工、磷酸化作用、異戊二烯化、外消旋作用、硒化作用、硫酸化作用、t-RNA介導的將胺基酸加入到蛋白質的過程如精氨醯化。(參考Creighton，T.E.，Proteins-structure and Molecular Properties第二版，W.H.Freeman andCompany，New York(1993)；Posttranslational Covalent Modification of Proteins，B.C.Johnson編著，Academic Press，NewYork，1-12頁(1983))。
正如此處所用，術語「分離的」指物質離開其原始環境(例如，如果是天然存在的，則離開其天然環境)。例如，存在於活的動物體內的天然存在的多核苷酸或多肽不是分離的，但從天然系統中的一些或全部共存物質中分離出的相同多核苷酸或多肽是分離的。這樣的多核苷酸可以是載體的一部分，和/或這樣的多核苷酸或多肽可以是組合物的一部分，它們仍然是分離的，原因在於這些載體或組合物不是天然環境的一部分。
正如此處所用，術語「純化的」不要求完全純化；更確切地說，該術語是一個相對定義。
正如此處所用，術語「重組的」指核酸與「骨架」核酸相鄰，而在其天然環境中與「骨架」核酸不是相鄰的。根據本發明的骨架分子包括核酸，如表達載體、自我複製核酸、病毒、整合核酸和其它用於維持或操縱相關核酸插入物的載體或核酸。
「重組的」多肽或蛋白質指通過重組DNA技術產生的多肽或蛋白質；即，通過編碼期望多肽或蛋白質的外源DNA構建物轉化的細胞產生的多肽或蛋白質。「合成的」多肽或蛋白質是那些通過化學合成製備的多肽或蛋白質。也可以固相化學肽合成方法來合成本發明的多肽或片段。這些方法自從20世紀60年代早期在本技術領域就已經已知(Merrifield，R.B.，J.Am.Chem.Soc.，852149-2154，1963)(也可以參見Stewart，J.M.和Young，J.D.，Solid Phase Peptide Synthesis，第二版，PierceChemical Co.，Rockford，I11，11-12頁))，並且最近已經被用於可通過商業途徑獲得的實驗室肽設計和合成試劑盒(Cambridge Research Biochemicals)。這些可通過商業途徑獲得的實驗室試劑盒通常使用了H.M.Geysen等人，Proc.Natl.Acad.Sci.，USA，813998(1984)的教導，在大量「棒」或「針」的尖端上提供合成肽，所有棒或針與單個板子相連接。當使用這樣的系統時，一個具有棒或針的板子被翻轉，且插入到具有相應孔或容器的第二個板子中，所述孔或容器含有將適當胺基酸附著或固定到針或棒尖端的溶液。通過重複這一處理步驟，即將棒或針的尖端翻轉且插入到適當溶液中，胺基酸就被構建到期望肽中。此外，可以得到數量眾多的FMOC肽合成系統。例如，多肽或片段的裝配可以使用Applied Biosystems，Inc.提供的Model 431A自動肽合成儀在固體載體上進行。該肽合成儀提供了現成獲取本發明所述肽，或者通過直接合成或者通過合成一系列片段，這些片段可以使用其它已知技術偶聯。
當起動啟動子轉錄的RNA聚合酶將編碼序列轉錄到mRNA中時，啟動子序列與編碼序列是「可操縱連接」的。
「質粒」由前面的小寫字母「p」，和/或其後的大寫字母和/或數字標明。此處的起始質粒或者通過商業途徑獲得，是不受限制地公開獲得，或者可以用與公開方法一致的方法從可獲得的質粒構建而來。另外，與此處所描述的質粒等效的質粒在本領域是已知的，對於普通技術人員而言是明了的。在本發明中，構建了一些典型的質粒，參考附圖2。這些質粒是實施本發明的一種形式。
「寡核苷酸」或者指一個單鏈多脫氧核苷酸，或者指兩個互補的多脫氧核苷酸鏈，它們可以化學合成。這樣化學合成的寡核苷酸沒有5』磷酸，因此在沒有用ATP添加磷酸和存在激酶的情況下，不會與另一個寡核苷酸連接。合成寡核苷酸將與沒有發生去磷酸化作用的片段連接。
正如此處所用，「片段」是天然存在的蛋白質的一部分，其可以以至少兩種不同的構象存在。片段可以具有與天然蛋白質相同或基本上相同的胺基酸序列。「基本上相同」指胺基酸序列大部分是相同的，但不是完全相同。
術語「變體」指在一個或多個鹼基對、密碼子、內含子、外顯子或胺基酸殘基(分別地)被修飾而不同於本發明的「母序列或者參考序列」。變體可以通過很多方法產生，這些方法包括，例如易錯PCR、改組、寡核苷酸介導的誘變、裝配PCR、有性PCR誘變、體內誘變、盒式誘變、遞歸集團誘變(recursive ensemblemutagenesis)、指數集團誘變(exponential ensemble mutagenesis)、位點特異性誘變、基因再裝配、GSSM及其任意組合。
從又一個方面來說，本發明所述的「子序列或者衍生序列」可以看作「母序列或者參考序列」的「變體」。因為它們在結構上與抗原中的「母序列或者參考序列」具有一定的相似性，而且又具有一定的差異。所以含有「子序列或者衍生序列」的物質可以誘發機體針對該抗原的免疫反應，尤其可以打破機體對該抗原業已產生的耐受性。其實，真是由於本發明所述疫苗中所用物質與抗原結構有一定相同性，機體發動的針對本發明疫苗中的物質的免疫反應就會交叉作用於該抗原。同時因為本發明所述疫苗中所用物質與抗原結構有一定差異，所以本發明的疫苗特別適用於誘發機體針對產生耐受性的抗原進行反應。
以下以EGFR分子疫苗為例來說明本發明的方法。事實上，本發明並不限於EGFR分子疫苗，本技術領域的技術人員根據本發明的教導可以方便地將本發明的方法用於其他疫苗的製備中。本發明更重要的價值在於提出了一種製備疫苗，特別是打破機體耐受性的方法；尤其在於腫瘤治療上有重要價值。
EGFR分子疫苗就是利用經現代生物工程技術改造進化的或在生物進化過程中形成的來源於人或其他異種生物的同源EGFR分子作為抗原而構建的各種新生物技術疫苗，包括重組蛋白疫苗、重組基因疫苗、重組病毒疫苗、基因修飾疫苗和穩定轉化共生菌。這是一種新型概念的腫瘤疫苗。
同源EGFR分子就是指與人EGFR分子在胺基酸或核苷酸水平上存在不同程度的相同程度的其它物種來源(如小鼠、大鼠、雞、鮐、果蠅等，見SEQ ID NO 1-14)或人工進化的EGFR分子的總稱。
人表皮生長因子受體(epidermal growth factor receptor，EGFR)是一種分子量為170KD的跨膜單鏈糖蛋白，由1186個胺基酸組成，具有酪氨酸蛋白激酶(PTK)活性。EGFR由胞外配體結合區、跨膜疏水區、近膜區、PTK活性區和受體C端尾肽五個部分組成。EGFR有多種不同形式的轉錄方式(見SEQ ID NO 1-5)，還有多種三種突變體，其中主要的突變體是第III類EGFRvIII(又稱ΔEGFR或de2-7EGFR，見SEQ ID NO 6)，是惡性腫瘤細胞膜上所特有的，具有高度的腫瘤特異性。人EGFR分子是一類基因家族，除EGFR(亦稱c-erbB1/HER1)外其它家族成員還包括c-erbB2/HER2、c-erbB3/HER3和c-erbB4/HER4，它們分別編碼膜蛋白p185erbB2、p160 erbB3、p180 erbB4。雖然這些成員具有高度同源的胺基酸序列和相似的結構特徵，但配體各不相同。本發明所涉及的腫瘤疫苗靶分子是EGFR(c-erbB1/HER1)，不包括c-erbB2/HER2、c-erbB3/HER3和c-erbB4/HER4。
EGFR分子的配體EGF或TGF-可通過自分泌(autocrine)或旁分泌(paracrine)等途徑作用於EGFR，使其PTK活化，經過一系列的信號傳遞，繼而導致細胞的分裂增殖。
EGFR廣泛分布於正常的哺乳動物上皮細胞表面，平均每個細胞的受體數為5-10萬。多種腫瘤細胞如肺癌、乳腺癌、卵巢癌、結腸癌、前列腺癌、胃癌、膀胱癌、頭頸部鱗癌和膠質瘤等，都過量表達EGFR，數量可達1-3×106/細胞。以肺癌為例，EGFR的過量表達還與肺癌的浸潤、轉移和預後密切相關。因此，EGFR被認為是腫瘤治療的一個理想的靶分子。目前，以EGFR為靶分子的治療，主要表現為EGFR單克隆抗體和小分子化合物。此外，以EGFRvIII為靶分子的腫瘤肽疫苗、以及利用反義核酸技術進行基因治療等取得了一些有益的進展。以EGFR分子為靶分子設計抗腫瘤分子疫苗，目前尚無任何文獻報導和專利報告。
腫瘤細胞在其惡性轉化、增殖的過程中要產生一種或多種腫瘤抗原。然而，在大多數情況下，腫瘤抗原的免疫原性較弱，並不足以引起機體的主動免疫反應。從免疫學的角度看，腫瘤細胞就是一種能不斷表達「正常」抗原(基因過度表達)和/或「異常」抗原(基因修飾、突變或缺失等)的宿主體內「自身」組織細胞，因此腫瘤抗原可看作是自體抗原。在正常情況下，機體對自體抗原不產生免疫應答，即呈現出免疫耐受。事實上，自體抗原是宿主免疫系統必須耐受的最相關和最豐富的抗原。自體抗原耐受的誘導和維持是由多種機制介導的，它們能防止對正常組織的不適當破壞。然而，當機體隱蔽的抗原釋放、或生物、物理與化學等因素使自體抗原發生改變等情況下，卻可導致自體免疫反應，造成靶抗原所在的細胞、組織或器官的病理性損傷和功能障礙。這樣，如果能使腫瘤細胞隱蔽的自體抗原釋放或者使其發生某種改變的話，則可能因此誘導出機體對自身腫瘤細胞的特異性自體免疫反應，從而使腫瘤縮小乃至消退。如前文所述，EGFR分子在多種腫瘤中過量表達，因此，EGFR分子可看作是一種腫瘤抗原，也是一種自體抗原。因此，可對人EGFR抗原分子(見SEQ ID NO 1-6)進行各種生物技術改造，製備成分子疫苗，從而誘導抗腫瘤免疫反應，達到抗腫瘤作用的目的。
生物在進化過程中，不同物種之間存在著具有不同程度同源性的大量分子，如EGFR分子就廣泛存在於人、鼠、果蠅、魚類、鳥類等生物，它們相互之間存在不同的同源性程度，人的EGFR分子在胺基酸水平上與鼠、雞和果蠅的EGFR分子的同源性分別為90％、64％和32％。我們可以利用異種同源分子在進化過程中所形成的細微差別來打破免疫耐受、增強免疫原性、誘導腫瘤細胞的自體免疫反應進而達到抗腫瘤的目的。這其中的機制很可能是由於在進化過程中同源基因的中性突變雖不導致其功能的喪失或改變，但卻可能影響到或改變了其免疫應答的方式。當異種同源基因導入到受試對象體內表達出相應的異種同源蛋白，受試者將之識別為外來抗原，一方面產生相應的抗體或細胞毒性淋巴細胞來清除它，另一方面由於這些表達出的異種同源蛋白與受試者體內相應的蛋白分子存在某種程度上的差異而與之產生非特異性的交叉反應，從而誘導自身免疫反應，打破機體對自身的這種蛋白的免疫耐受(見說明書附圖1)。
如前文所述，人的EGFR與其他物種(如鼠、雞、果蠅等)的EGFR在胺基酸水平上表現出不同程度的同源性，通過BLAST分析發現人的EGFR在胺基酸水平上與鼠、雞和果蠅的同源性分別90％、64％和32％。我們利用異種同源EGFR分子產生的種與種之間的免疫交叉反應來誘導機體的主動免疫反應，克服自身腫瘤細胞的免疫耐受，通過其產生的抗體競爭性抑制EGFR與配體EGF或TGF-的結合，從而抑制腫瘤細胞的生長，此外還通過其所誘導的CTL來抑制腫瘤細胞的生長。
此外，現代生物工程技術的發展，如易錯PCR技術、DNA「洗牌術」(DNAshuffling)、噬菌體展示技術等，為EGFR分子的人工基因直接進化(gene directedevolution)提供了技術支持。
本發明涉及抗腫瘤EGFR分子疫苗，包括自體EGFR分子疫苗(autologousEGFR vaccine)、異種EGFR分子疫苗(xenogeneic EGFR vaccine)和基因直接進化EGFR分子疫苗(directed evolution EGFR Vaccine)。
本發明主要涉及一種新型的腫瘤疫苗——EGFR分子疫苗，它是利用經現代生物工程技術改造的或在生物進化過程中形成的來源於人或其他異種生物的同源EGFR分子作為抗原而構建的各種新生物技術疫苗，包括蛋白疫苗、基因疫苗、病毒疫苗、基因修飾疫苗和穩定轉化共生菌。EGFR分子疫苗對各種過表達EGFR分子的實體瘤都具有抗腫瘤作用效應，其抗腫瘤作用機制在於EGFR分子作為免疫交叉抗原，能打破機體對自身EGFR分子的免疫耐受，誘導機體產生針對EGFR分子自身免疫樣交叉反應。抗腫瘤EGFR分子疫苗也可以是利用納米生物技術製備而成的納米粒製劑。
一、EGFR分子的來源、選擇與改造本發明涉及的EGFR分子包括自體EGFR分子(autologous EGFR molecule)和異種EGFR分子(xenogeneic EGFR molecule)和基因直接進化EGFR分子(directedevolution EGFR molecule)。
利用在生物進化過程中形成的或經現代生物工程技術改造的同源EGFR分子的差異性進行抗腫瘤免疫治療是本發明的最大特徵。
EGFR在自然界中分布廣泛，從哺乳動物(如人、鼠等)、鳥類(如雞等)、魚類(如鮐等)到低等生物(如線蟲、果蠅等)都有EGFR的表達，這些生物來源的EGFR分子之間都存在一定的差異，其同源性程度在30-100％之間。本發明所涉及的異種EGFR分子的同源性在30-95％之間，主要涉及的生物物種是人、鼠、雞和果蠅，在胺基酸水平上，人的EGFR分子與鼠、雞和果蠅的EGFR分子的同源性分別為90％、64％和32％，人、鼠、雞和果蠅的EGFR分子分別代表了本發明中所涉及的同源性程度的監測點。
自體EGFR分子由於存在免疫耐受，故免疫原性較弱。利用現代生物工程技術可對自體EGFR分子進行人工直接進化(gene directed evolution)和改造，增強EGFR分子作為抗原的免疫原性。通常是利用易錯PCR、隨機引物延伸技術和DNA「洗牌」術(DNA shuffling)等對自體EGFR分子進行人工突變，建立基因突變庫，然後使用噬菌體展示技術(phage display)、核糖體展示技術(ribosome display)等進行篩選，從而得到免疫原性強的EGFR分子。
此外，利用細菌、病毒等不同生物的表達修飾系統的差異，也可以在蛋白質水平上對自體EGFR分子進行人工改造，增強自體EGFR的免疫原性。
二、EGFR重組基因疫苗基因疫苗是利用現代分子生物學技術構建製備的以核酸為基礎的新型疫苗，主要是DNA疫苗。
本發明的EGFR分子疫苗之一就是DNA疫苗。依據GenBank等各公開的資料庫所收藏的各種生物來源(如人、小鼠、大鼠、雞、鮐、果蠅等，見SEQ ID NO1-14)的EGFR分子的序列(包括基因、cDNA、mRNA和胺基酸序列)按常規方法設計引物或探針，用PCR、RT-PCR、雜交等技術，從各種商品化的基因文庫、cDNA文庫或各種細胞系、組織等中克隆分離出不同種屬生物來源的EGFR分子的胞外區段的cDNA(發明人發現EGFR胞外區段才是引起免疫反應的反應域)，或進一步用基因直接進化技術篩選到的具有強免疫原性的EGFR分子。各種來源的EGFR cDNA的胞外段序列經測序鑑定後，用分子克隆技術構建其真核生物表達質粒系統，然後轉染入CHO細胞系，觀察並檢測其表達EGFR的情況和水平。各重組EGFR分子的真核生物表達質粒可經過限制性酶切分析、SDS-PAGE和Western Blot等進行鑑定、確證。用鹼法抽提經過鑑定的重組EGFR分子表達質粒，用超離心、超濾等方法去除大腸桿菌內毒素，即得到純淨的質重組粒DNA，這些質粒DNA即可作為DNA疫苗進行免疫。
DNA分子疫苗的代表性質粒圖譜如圖2A所示，其具體構建過程簡要敘述如下依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GACCATGGAGGAAAAGAAAGTTTGC 3』，5』ACGAATTCTTAGGACGGGATCTTAGGCCCA3』；小鼠的引物為5』GACCATGGAGGAAAAGAAAGTCTGC 3』，5』ACGAATTCTTAATAGATGGTATCTTTGGC 3』；雞的引物為5』GACCATGGAGGAGAAGAAAGTTTGTC 3』，5』ACGAATTCTTAAGATGGAGTTTTGGAGCC 3』)，分別以人肺癌細胞株A431、小鼠肺癌細胞株LL2和雞胚的總RNA為模板進行RT-PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用NcoI和EcoRI酶切，收集1.9kb片段並純化，插入到用NcoI和EcoRI雙酶切的pORF-MCS(InvivoGen公司)載體，篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為pORF-hEGFR、pORF-mEGFR和pORF-chEGFR。至於pcDNA-hEGFR、pcDNA-mEGFR和pcDNA-chEGFR的構建過程與上述方法一致(pcDNA3.1(+)載體來源於Invitrogen公司)，只是PCR引物略有改動，分別是人的引物5』GAGCTAGCATGGAGGAAAAGAAAGTTTGC 3』，5』CACTCGAGTTAGGACGGGATCTTAGGCCCA 3』；小鼠的引物為5』GAGCTAGCATGGAGGAAAAGAAAGTCTGC 3』，5』CACTCGAGTTAATAGATGGTATCTTTGGC 3』；雞的引物為5』GAGCTAGCATGGAGGAGAAGAAAGTTTGTC 3』，5』CACTCG AGTTAAGATGGAGTTTTGGAGCC 3』。
用人的EGFR基因胞外區段構建和製備DNA疫苗免疫Lewis肺癌的模型小鼠，發現免疫後的第8周，注射人EGFR疫苗的小鼠生存率為78％，明顯高於注射過小鼠EGFR疫苗(25％)和對照實驗動物(10％～15％)，同時並未發現模型小鼠的肺、肝、心、腎等有病理性改變，進一步的研究表明誘導的自體免疫反應主要依賴於CD4+T淋巴細胞，CTL活性檢測沒有發現靶細胞特異性的殺傷效應。免疫組化結果顯示腫瘤組織有自身抗體沉著，而肺、肝等組織則沒有，自身反應抗體主要為IgG。
以EGFR分子為模板設計的各種反義RNA和RNAi可看作是EGFR重組基因疫苗的特例，它主要不是通過提高EGFR分子的免疫原性，誘導產生抗EGFR抗體和特異性的CTL，進而阻斷EGFR信號通路，誘導腫瘤細胞凋亡，抑制腫瘤細胞的生長和擴散，而是在DNA和RNA水平上直接抑制和阻斷EGFR分子的表達。
三、EGFR重組蛋白疫苗蛋白疫苗是比較傳統形式的疫苗，但蛋白質有較好的免疫原性。本發明的EGFR分子疫苗之一就是重組蛋白疫苗，包括以各種大腸桿菌重組表達載體、酵母重組表達載體、棒狀病毒重組表達載體等表達系統表達出的重組蛋白所構建而成的疫苗。
利用分子克隆技術(如PCR、RT-PCR、雜交等技術)從各種商品化的基因文庫、cDNA文庫或各種細胞系、組織等中克隆分離出不同種屬生物來源的EGFR分子的胞外區段的cDNA，或進一步用基因直接進化技術篩選到的具有強免疫原性的EGFR分子，然後用分子克隆技術構建其原核生物表達質粒系統，轉化合適的大腸桿菌宿主，觀察並檢測其表達EGFR的情況和水平。各重組EGFR分子的表達質粒可經過限制性酶切分析、SDS-PAGE和Western Blot等進行鑑定、確證。經確證的重組EGFR分子原核表達質粒轉化E.coli，大量培養重組菌落，低溫離心收集菌體，菌體經PBS重懸後用超聲法破碎細胞，用離子交換層析、親和層析等方法分離純化重組EGFR蛋白，此重組的EGFR蛋白質即可作為蛋白疫苗進行免疫。
E.coli表達的重組EGFR蛋白疫苗的代表性質粒圖譜如圖2B所示，其具體構建過程簡要敘述如下依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GACCATGGAGGAAAAGAAAGTTTGC 3』，5』ACAGATCTAGGACGGGATCTTAGGCCCA 3』；小鼠的引物為5』GACCATGGAGGAAAAGAAAGTCTGC 3』，5』ACAGATCTATAGATGGTATCTTTGGC 3』；雞的引物為5』GACCATGGAGGAGAAGAAAGTTTGTC 3』，5』ACAGATCTAGATGGAGTTTT GGAGCC 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用NcoI和BglII酶切，收集1.9kb片段並純化，插入到用NcoI和BglII雙酶切的pQE60(QIAGEN公司)載體，篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為pQE-hEGFR、pQE-mEGFR和pQE-chEGFR。
除上述利用大腸桿菌重組表達系統表達各種來源的EGFR分子外，還可以利用酵母重組表達系統、棒狀病毒重組表達系統等表達EGFR重組蛋白，從而製備重組蛋白疫苗。EGFR重組蛋白疫苗的構建流程圖見圖3。
酵母表達的重組EGFR蛋白疫苗的代表性質粒圖譜如圖2C所示，其具體構建過程簡要敘述如下依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』ATACTCGAGAAAAGAGAGCTGGAGGAAAAGAAAG 3』，5』GCTCTAGAATGGCACAGGTGGCACA 3』；小鼠的引物為5』ATGCTCGAGAAAAGAGAGTTGGAGGAAAAGAAAGTC 3』，5』AAGCGGCCGCCATAGATGGTATCTTTG 3』；雞的引物為5』ATACTCGAGAAA AGAGAGGTGGAGGAGAAGAAAG3』，5』CGTCTAGAAGATGGAGTTTTGGAG 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用XhoI和XbaI(對小鼠的克隆為XhoI和NotI)酶切，收集1.9kb片段並純化，插入到用XhoI和XbaI雙酶切(對小鼠的克隆為XhoI和NotI雙酶切)的pPICZ A(Invitrogen公司)載體，轉化E.coli篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為酵母表達質粒pYE-hEGFR、pYE-mEGFR和pYE-chEGFR。這些酵母表達質粒經PmeI酶切線性化後，用電穿孔法轉化酵母菌株X33或GS115，用Zeocin抗性篩選穩定轉化子，在MMH(Minimal Methanolwith histidine，MMH)和MDH(Minimal Dextrose with histidine，MDH)瓊脂平板上鑑定和挑選Mut+轉化子。挑選6-10個Mut+轉化子進行小規模表達，用SDS-PAGE、Western Blot、ELISA等方法對所表達的重組蛋白進行鑑定。選擇其中表達效率最高的Mut+轉化子做大規模表達，建立各級酵母表達種子庫。重組菌株大量搖瓶培養或發酵，低溫離心收集菌體，菌體經PBS重懸後用超聲法破碎細胞，用離子交換層析、親和層析等方法分離純化重組EGFR蛋白，此重組的EGFR蛋白質即可作為蛋白疫苗進行免疫。
同樣地，也可用其他的酵母表達系統構建類似的酵母重組表達質粒。
重組蛋白疫苗具有比DNA疫苗更強的誘導免疫交叉反應的作用，產生高滴度的抗EGFR抗體以及特異性的CTL，從而抑制腫瘤細胞的生長和轉移。
四、EGFR重組病毒疫苗重組病毒也是一種良好的分子疫苗轉運系統，這些重組病毒表達系統構建的分子疫苗包括重組腺病毒疫苗、Lentivirus疫苗、腺病毒相關病毒疫苗、逆轉錄病毒疫苗、痘苗病毒疫苗和單純皰疹病毒疫苗。
腺病毒載體是目前在腫瘤基因治療中最有效的載體之一，它具有滴度高、安全性好、能感染分裂或不分裂細胞、不整合插入染色體等優點。此外，腺病毒還具有較強的免疫原性，這對於基因治療也許是一個缺點，但在基因免疫治療中卻可能是一個很大的優點。重組腺病毒疫苗是EGFR重組病毒疫苗最重要的一種。同前文所述，首先克隆出各種同種、異種或基因直接進化的EGFR cDNA，然後用分子生物學技術構建其重組腺病毒表達載體，轉染293細胞，即得到重組的腺病毒。重組的腺病毒經PCR、Western Blot等加以確證。利用293細胞大量擴增經確證的EGFR重組腺病毒疫苗，用超離心、超濾等技術分離、純化重組腺病毒，此經純化的EGFR重組腺病毒即可作為疫苗進行免疫。EGFR重組腺病毒疫苗的構建流程示意圖見圖4。
EGFR重組腺病毒是利用AdEasy系統構建而成的，其具體構建過程簡要敘述如下依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GAAGATCTATGGAGGAAAAGAAAGTTTGC 3』，5』ACGATATCTTAAGGACGGGATCTTAGGCCCA 3』；小鼠的引物為5』GAAGATCTATGGAGGAAAAGAAAGTCTGC 3』，5』ACGATATCTTAATAGATGGTATCTTTGGC 3』；雞的引物為5』GAAGATCTATGGAGGAGAAGAAAGTTTGTC 3』 ，5』ACGATATCTTAAGATGGA GTTTTGGAGCC 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用BglII和EcoRV酶切，收集1.9kb片段並純化，插入到用BglII和EcoRV雙酶切的pShuttle-CMV(Quantum Biotechnologies公司)載體，篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為腺病毒穿梭表達質粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR(圖2D)。分別將經PmeI酶切的腺病毒穿梭表達載體pShuttle-EGFR與包含腺病毒基因組的骨架載體pAdEasy-1或pAdEasy-2共轉化E.coli BJ5183，得到重組腺病毒載體質粒pAd-hEGFR、pAd-mEGFR和pAd-chEGFR。這些重組腺病毒載體質粒經PacI酶切後，用磷酸鈣-DNA共沉澱法轉染入腺病毒包裝細胞株293細胞中，得到相應的重組腺病毒Ad-hEGFR、Ad-mEGFR和Ad-chEGFR。用PCR、Western blot等確證EGFR基因構建入重組腺病毒載體中並能在真核細胞中得到正確有效的表達。
根據EGFR重組腺病毒疫苗中所包含腺病毒基因組的不同，又有EGFR重組I代腺病毒(腺病毒穿梭表達質粒pShuttle-EGFR與pAdEasy-1的重組體，命名為Ad-hEGFR I、Ad-mEGFR I和Ad-chEGFR I)疫苗和EGFR重組II代腺病毒(腺病毒穿梭表達載體pShuttle-EGFR與pAdEasy-2的重組體，命名為Ad-hEGFR II、Ad-mEGFR II和Ad-chEGFR II)疫苗之分。
此外，EGFR重組腺病毒疫苗還可以用甘露聚糖(Mannan)等進行靶向修飾。
EGFR重組腺病毒疫苗由於腺病毒良好的基因導入性，能有效誘導機體產生抗腫瘤免疫反應，抑制EGFR高表達腫瘤的生長。
Lentivirus病毒載體是新一代的基因治療載體，它來源於HIV-1的複製缺陷型lentivirus，與傳統的來源於的莫洛尼氏白血病毒(Moloney Leukemia Virus，MoMLV)的逆轉錄病毒載體不同，它對處於分裂和不分裂狀態的哺乳動物細胞都能進行有效轉染，且生物安全性更好。此外，與腺病毒載體不同的是，Lentivirus病毒載體能整合到細胞染色體中，從而使導入的外源基因獲得穩定、長久的表達。重組Lentivirus病毒疫苗也是EGFR重組病毒疫苗較重要的一種。同前文所述，首先克隆出各種同種、異種或基因直接進化的EGFR cDNA，然後用分子生物學技術構建其重組Lentivirus表達載體，轉染293FT細胞，即得到重組的Lentivirus病毒。重組的Lentivirus病毒經PCR、Western Blot等加以確證。利用293FT細胞大量擴增經確證的EGFR重組Lentivirus病毒疫苗，用超離心、超濾等技術分離、純化重組Lentivirus病毒，此經純化的EGFR重組Lentivirus病毒即可作為疫苗進行免疫。EGFR重組Lentivirus病毒疫苗的構建流程示意圖見圖4B。
EGFR重組Lentivirus病毒是利用ViraPowerTMLentiviral GatewayExpressionKit(Invitrogen公司)構建而成的，其具體構建過程簡要敘述如下依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GACCATGGAGGAAAAGAAAGTTTGC 3』，5』ACGATATCAGGACGGGATCTTAGGCCCA 3』；小鼠的引物為5』GACCATGGAGGAAAAGAAAGTCTGC 3』，5』ACGATATCATAGATGGTATCTTTGGC 3』；雞的引物為5』GACCATGGAGGAGAAGAAAGTTTGTC 3』，5』ACGATATCAGATGGAGTTTTGGAGCC 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用NcoI和EcoRV酶切，收集1.9kb片段並純化，插入到用NcoI和EcoRV雙酶切的pENTR11(Invitrogen公司)載體中，篩選重組質粒，命名為pENTR-hEGFR、pENTR-mEGFR和pENTR-chEGFR(圖2E)。分別將這些載體pENTR-EGFR與包含Lentivirus病毒基因組的骨架載體pLenti6/V5-DEST共轉化E.coli DH5，得到重組Lentivirus病毒載體質粒pLenti-hEGFR、pLenti-mEGFR和pLenti-chEGFR(圖2E)。這些重組Lentivirus病毒載體質粒與包裝混合物一起(ViraPowerTMPackagingMix)用磷酸鈣-DNA共沉澱法轉染入Lentivirus病毒包裝細胞株293FT細胞中，得到相應的重組Lentivirus病毒Lenti-hEGFR、Lenti-mEGFR和Lenti-chEGFR。用PCR、Western blot等確證EGFR基因構建入重組Lentivirus病毒載體中並能在真核細胞中得到正確有效的表達。
五、EGFR分子疫苗的修飾和改進前文的重組EGFR分子疫苗(包括重組EGFR基因疫苗、蛋白疫苗和病毒疫苗)只是基本形式，本發明還在高效性、特異性等方面有許多修飾和改進，這些改進包括1.EGFR分子疫苗佐劑的選擇合適的佐劑是有效提高疫苗的免疫效應的良好載體，不同的疫苗形式有不同的佐劑。本發明EGFR DNA疫苗的佐劑主要是福氏佐劑和脂質體，EGFR蛋白疫苗的佐劑主要是鋁鹽，EGFR重組病毒疫苗通常不使用佐劑。
2.EGFR基因修飾疫苗基因修飾疫苗是指轉染了人或其他異種生物的EGFR分子的細胞疫苗，包括使用穩定轉化的各種腫瘤細胞株、血管內皮細胞和樹突狀細胞製備的細胞疫苗、用各種重組病毒感染的腫瘤細胞或腫瘤組織製備的疫苗。EGFR基因修飾疫苗是EGFR分子疫苗的一種特例形式。
前文已描述多種腫瘤包括肺癌、乳腺癌、卵巢癌、結腸癌、前列腺癌、胃癌、膀胱癌、頭頸部鱗癌和膠質瘤等都高表達EGFR，但這些腫瘤均對EGFR耐受，並不產生抗EGFR的免疫反應。但是，利用基因修飾疫苗可打破免疫耐受，產生抗EGFR免疫反應。用重組的各種來源的EGFR分子的真核表達質粒轉染各種腫瘤細胞株(如肺癌細胞株A431、乳腺癌細胞株MCF7等)、腫瘤血管內皮細胞等，篩選穩定表達EGFR分子的轉化株，收穫穩定轉化株，用副甲醛固定法等製備細胞疫苗。此外，也可以用各種來源的EGFR構建的各種EGFR重組病毒疫苗感染各種腫瘤細胞株、腫瘤組織和腫瘤血管內皮細胞等，用副甲醛固定法等製備細胞疫苗。
3.EGFR穩定轉化共生菌EGFR分子疫苗還有活體疫苗形式，這就是EGFR穩定轉化共生菌。
用各種來源的(包括自體的、異體的和基因直接進化的)EGFR構建的原核生物重組表達質粒，轉化腸道共生菌，如雙岐桿菌等，篩選其穩定表達菌株。這些穩定表達的腸道共生菌菌株能持續分泌外源EGFR分子，誘導機體免疫反應，是一種活體形式的EGFR疫苗，它是EGFR分子疫苗的特例。
4.EGFR分子疫苗的靶向納米粒製劑單純的重組EGFR分子疫苗(包括重組EGFR基因疫苗、蛋白疫苗和病毒疫苗)依然有它的缺陷，其特異性不夠，免疫原性仍有待提高。本發明利用納米生物技術對EGFR分子疫苗進行靶向修飾，製備EGFR分子疫苗的靶向納米粒製劑。這些靶向納米粒以EGFR分子(包括DNA和蛋白質形式)為抗原靶分子，以生物性分子如脂質體、可降解性高分子生物材料如聚丙交酯-乙交酯(polyDL-lactide-co-glycolide polymer，PLGA)等為納米材料，以MIP-3、MIP-3等基因以及甘露聚糖(Mannan)、Flt3-L等為修飾基團，以樹突狀細胞為靶細胞構建而成。
本發明的EGFR分子疫苗靶向納米粒製劑有多種形式，主要包括①甘露糖化納米粒(包括甘露糖化腺病毒重組EGFR疫苗、甘露糖化重組EGFR蛋白疫苗、甘露糖化脂質體重組EGFR基因疫苗、甘露糖化重組EGFR蛋白疫苗等)，②基因靶向納米粒(基因靶向納米脂質體重組EGFR疫苗、基因靶向納米PLGA重組EGFR疫苗、基因靶向重組EGFR腺病毒疫苗等)，它們同時表達EGFR和MIP-3等基因，③腫瘤新生血管靶向腺病毒重組EGFR疫苗(RGD修飾的腺病毒重組EGFR疫苗)。
本發明的納米粒粒徑通常在500nm以下，可分為三種規格200-500nm、100-200nm和50-100nm，以50-100nm粒徑的納米粒的效果為最佳，納米粒峰值在80nm左右。
甘露糖化腺病毒重組EGFR疫苗的製備過程簡要描述如下用常規方法擴增EGFR重組腺病毒(I代、II代均可)，層析或超離心法純化重組腺病毒。將70mgmannan(sigma)溶於5ml 0.1M的磷酸鹽緩衝液(pH6.0)中，終濃度14mg/ml，加45ml 0.01M高碘酸鈉溶液，在4℃下混合氧化60分鐘，加入10l乙二醇，在4℃下孵育30分鐘，即得Ox-M(Oxidative Mannan，Ox-M)混合物。將Ox-M混合物倒入用重碳酸鹽緩衝液(pH6.0-9.0)平衡Sephadex-G25層析柱進行層析分離，OX-M即被洗入2ml的空容器。將純化的Ox-M與1×1014腺病毒顆粒混合，室溫過夜，即獲得所需Ox-M-腺病毒。在Ox-M-腺病毒中加入1mg/ml硼氫化鈉，室溫放置3小時即得Red-M-腺病毒(Reductive Mannan，Red-M)。Ox-M-腺病毒與Red-M-腺病毒經超濾脫鹽、濃縮後，過濾細菌，小管分裝，-80℃低溫保存。此經純化的甘露糖化重組EGFR腺病毒即可作為疫苗進行免疫。
本發明認為腺病毒顆粒的粒徑在80nm左右，是天然的納米粒。對腺病毒顆粒進行修飾，使腺病毒纖維蛋白(Adenoviral fiber protein)表達RGD三肽，該三肽對腫瘤血管內皮細胞有特異靶向性。用RGD修飾的腺病毒重組EGFR疫苗，可看作是天然的靶向納米EGFR分子疫苗。
本發明利用AdEasy系統構建RGD修飾腺病毒的重組EGFR疫苗，圖5顯示了RGD修飾的腺病毒重組EGFR疫苗的構建流程，其具體過程是腺病毒基因組骨架質粒pAdEasy-1和pAdEasy-2經限制性內切酶SpeI(Sp)酶切後，用T4 DNA聚合酶補平(filling，f)末端，再用PacI(P)酶切，分別電泳回收6211bp和3579bp的片段，分別命名為AdFiber I/Sp/f/P和AdFiber II/Sp/f/P，該片段包含了完整的腺病毒纖維蛋白基因。把AdFiber I/Sp/f/P和AdFiber II/Sp/f/P片段插入經BamHI酶切-T4 DNA聚合酶補平-PacI酶切處理(BamHI/filling/PacI-digested)的pShuttle載體，將所得的重組質粒分別命名為pSh-AdFiber I和pSh-AdFiber II。pSh-AdFiberI用NheI酶切-T4 DNA聚合酶補平-KpnI酶切處理(NheI/filling/KpnI)，電泳回收2090bp的片段AdFiber I/Nh/f/K，將該片段插入到經SmaI/KpnI雙酶切的pUC18載體中，所得的重組質粒命名為pUC-AdFiber I；而pSh-AdFiber II用AvrII酶切-T4 DNA聚合酶補平-HindIII酶切處理(AvrII/filling/HindIII)，電泳回收838bp的片段AdFiber I/A/f/H，將該片段插入到經SmaI/HindIII雙酶切的pUC18載體中，所得的重組質粒命名為pUC-AdFiberII。設計一系列PCR引物以便以pUC-AdFiberI和pUC-AdFiberII為模板擴增腺病毒疣足(Adenovirus knob，Ad-knob)基因序列，引物分別是F1(5』GAAAGCTAGC CCTGCAAACATCA 3』)、R1( 5』ACTCCCGGGAGTTGTGTCTCCTGTTTCCTG3』)、F2(5』ACTCCCGGGAGTGC ATACTCTATGTCA 3』)、R2(5』TATGGTACCGGGAGGTGGTGA 3』)、F3(5』AACCTAGGGAGGTTAACCTAAGCACTG 3』)、和R3(5』CTCAAGCTTTTTGG AATTGTTTGA 3』)。以引物F1-R1、F2-R2、F3-R1和F2-R3分別進行第一輪PCR，得到產物PCR1、PCR2、PCR3和PCR4，再以F1-R2和F3-R3為引物，以第一次擴增產物PCR1與PCR2、PCR3與PCR4為模板進行第二輪PCR擴增，得到PCR產物PCR1-PCR2(PCR I)、PCR3-PCR4(PCR II)，將第二輪PCR擴增的PCR I和PCR II插入到經EcoRV酶切的pBR322載體中，所得到的重組質粒命名為pBR-PCR I和pBR-PCR II。把RGD-4C雙螺旋寡聚核苷酸(RGD-4C duplex)5』TGTGACTGCCGCGGAGACTGTTTCTGC 3』3』ACACTGACGGCGCCTCTGACAAAGACG 5』插入到SmaI酶切的pBR-PCR I and pBR-PCR II載體中，將所得到的重組質粒命名為pBR-PCR/RGD I和pBR-PCR/RGD II，並對重組結構進行測序確證。用NheI/KpnI雙酶切pBR-PCR/RGD I，電泳回收PCR/RGD I片段，然後插入到NheI/KpnI雙酶切的pUC-AdFiber I載體中，所得的重組質粒命名為pUC-AdFiber-RGD I；用AvrII/HindIII雙酶切pBR-PCR/RGD II，電泳回收PCR/RGDII片段，再插入到AvrII/HindIII雙酶切的pUC-AdFiber II載體中，將所得到的重組質粒命名為pUC-AdFiber-RGD II。然後，用SpeI/PacI雙酶切pUC-AdFiber-RGDI和pUC-AdFiber-RGD II載體，電泳回收AdFiber-RGD I、AdFiber-RGD II片段，插入到SpeI/PacI雙酶切的pAdEasy-1、pAdEasy-2載體中，所得的重組質粒分別命名為pAdEasy-RGD.I、pAdEasy-RGD II。將經PmeI線性化的腺病毒穿梭質粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR分別與pAdEasy-RGD I和pAdEasy-RGD II共轉化E.coli BJ5183，所得的重組質粒命名為腺病毒質粒pAd-RGD-EGFR I和pAd-RGD-EGFR II，腺病毒質粒pAd-RGD-EGFR I轉染293細胞，所得的重組腺病毒命名為Ad-RGD-EGFR I，腺病毒質粒pAd-RGD-EGFR II轉染293E4pIX細胞，所得的重組腺病毒命名為Ad-RGD-EGFR II。經純化的Ad-RGD-EGFR I和Ad-RGD-EGFR II可作為疫苗進行免疫，對腫瘤血管內皮細胞具有特異的靶向性。
本發明發現納米靶向的EGFR分子疫苗能比各種常規的EGFR分子疫苗更有效地提高EGFR的免疫原性，誘導更強烈的抗腫瘤免疫反應。納米靶向EGFR分子疫苗抗腫瘤作用原理示意圖見圖6。
5.EGFR分子疫苗與其它免疫反應促進因子等的協同應用EGFR分子疫苗的抗腫瘤作用效應可在其它免疫促進因子的協同作用下得到增強，這些免疫促進因子包括各種細胞因子(cytokine，如IL-2、TNF-、IFN-、GM-CSF等)、各種趨化因子(Chemokine，如MIP3、MIP3、IP10等)、各種應急因子(如HSP70、HSP90等)、各種免疫刺激因子(如B7等)。各種免疫促進因子與EGFR分子疫苗的協同作用既可以通過基因融合在基因水平上實現，也可以通過蛋白質聯合應用在蛋白質水平上實現，還可以通過共轉染腫瘤細胞、樹突狀細胞和血管內皮細胞等在細胞水平上實現。
六、EGFR分子疫苗的抗腫瘤作用效應EGFR分子疫苗的重要生物學功能就是以同種、異種或直接進化的EGFR分子為抗原分子所構建的各種新生物技術疫苗，包括蛋白疫苗、基因疫苗、病毒疫苗和基因修飾疫苗等，都具有抗腫瘤作用，這些抗腫瘤作用表現為保護性抗腫瘤作用、治療性抗腫瘤作用以及抗腫瘤轉移作用。
為觀察EGFR重組DNA疫苗的抗腫瘤效應，給隨即分組的小鼠(每組15隻)分別肌肉注射100g的EGFR重組DNA疫苗hEe-p、mEe-p、c-p或生理鹽水，每周一次，連續四周。在第四次免疫結束後一周，給每隻免疫小鼠分別皮下接種5×105個LL/2c Lewis肺癌(圖7A和C)或MA782/5S乳腺癌細胞(圖7B和D)。從圖中可以看出，用mEe-p、c-p或生理鹽水免疫小鼠的腫瘤持續生長，而hEe-p免疫小鼠的保護性免疫效應則非常明顯，並且hEe-p免疫小鼠的生存率也明顯高於mEe-p、c-p或生理鹽水免疫小鼠，hEe-p免疫的小鼠生存期超過5個月，其在荷瘤150天後，接種LL/2c Lewis肺癌和MA782/5S乳腺癌的生存率分別有60％和66％。研究還發現，保護性免疫效應呈劑量依存效應，150g劑量的免疫效果與100g的免疫效果相當，而5-15g的劑量則幾乎沒有免疫效果。此外，hEe-p免疫EGFR陰性腫瘤(如H22肝癌和MMT-06052鼠乳腺癌)的荷瘤小鼠沒有保護性免疫效應。
除保護性免疫效應外，EGFR重組DNA疫苗還具有治療性免疫效應(圖8)。同樣地，給隨即分組的小鼠(每組15隻)首先皮下接種1 106個LL/2c Lewis肺癌(圖8A和C)或MA782/5S乳腺癌活細胞(圖8B和D)，5天後分別肌肉注射100g的hEe-p、mEe-p、c-p或生理鹽水，每周一次，連續四周。從圖中可以看出，用mEe-p、c-p或生理鹽水免疫小鼠的腫瘤持續生長，而hEe-p免疫小鼠的治療性免疫效應則非常明顯，並且hEe-p免疫小鼠的生存率也明顯高於mEe-p、c-p或生理鹽水免疫小鼠，hEe-p免疫的小鼠生存期超過5個月，其在荷瘤150天後，接種LL/2c Lewis肺癌和MA782/5S乳腺癌的生存率分別有40％和53％。
重組EGFR蛋白疫苗也同樣有保護性免疫效應和治療性免疫效應(圖9)。同前文所述，選擇6-8周齡雌性C57BL/6或BALB/c小鼠，隨機分組，建立LL/2c Lewis肺癌、MA782/5S乳腺癌和C26結腸癌荷瘤小鼠模型。荷瘤小鼠皮下注射重組蛋白疫苗5-50μg或佐劑氫氧化鋁磷膠及生理鹽水100μl，每周一次，連續四周。重組chEGFR蛋白疫苗免疫小鼠產生了明顯的抗腫瘤保護性免疫效應和治療性免疫效應，可抑制LL/2c Lewis肺癌和MA782/5S乳腺癌的生長，延長荷瘤小鼠的生存時間，但對EGFR陰性的C26腫瘤無明顯的影響。而用重組mEGFR蛋白疫苗、鋁佐劑免疫或注射生理鹽水等對照組的荷瘤小鼠的腫瘤則生長迅速，小鼠生存期明顯縮短。疫苗組腫瘤體積(t檢驗)和生存期(log-rank檢驗)與各對照組比較均有顯著差異(P＜0.05)。圖9就顯示了重組EGFR蛋白疫苗對MA782/5S乳腺癌荷瘤小鼠的抗腫瘤作用效果。
轉移是腫瘤進展以及放化療失敗的常見原因，腫瘤細胞在血液、淋巴循環中的存在以及微小轉移灶的形成是轉移的關鍵。本發明繼續觀察到重組EGFR分子疫苗還有抗腫瘤轉移作用(圖10)。在尾靜脈注射轉移模型的治療中發現，chEGFR蛋白疫苗免疫的荷瘤小鼠較少發生肺部轉移或程度遠較對照組輕，而重組mEGFR蛋白疫苗、鋁佐劑免疫或注射生理鹽水等對照組的荷瘤小鼠則100％出現肺部轉移，且轉移病灶的個數明顯為多，甚至無法計數。圖10顯示了LL/2c Lewis肺癌荷瘤小鼠經重組EGFR蛋白疫苗免疫的抗腫瘤轉移效應，從圖中可以看出，各組治療結束時，chEGFR蛋白疫苗免疫小鼠的LL/2c移植瘤肺部轉移灶數量和肺溼重均顯著較對照組為輕。
此外，EGFR分子疫苗還能抑制體外(in vitro)腫瘤細胞的生長及具有體內(invivo)過繼免疫抗腫瘤效應(圖11)。EGFR分子疫苗第四次免疫7天後，收集小鼠血清，用親和層析法純化血清中的免疫球蛋白(immunoglobulin，Ig)。向2×105/ml的處於對數生長期的EGFR陽性腫瘤細胞(A549，LL/2c，MA782/5S)和EGFR陰性腫瘤細胞(H22和MMT-06052)中加入不同濃度的Ig(1-1000mg/ml)共培養72小時，用臺盼藍染色法鑑定活細胞，計算細胞生長抑制率。結果顯示用人EGFR重組DNA疫苗hEe-p免疫小鼠來源的Ig處理的EGFR陽性腫瘤細胞呈現明顯的抑制生長狀態，而EGFR陰性的腫瘤細胞則沒有影響(圖11A)。作為對照，用未免疫的正常小鼠來源的Ig與相應的腫瘤共培養，無論對EGFR陽性腫瘤細胞還是EGFR陰性腫瘤細胞都沒有抑制作用(圖11B)。
這些純化的來源於免疫小鼠的Ig還具有過繼免疫作用效應。裸鼠皮下接種1×105-1×106個腫瘤細胞1天後，按10-300mg/kg靜脈輸注純化的Ig，每周二次，連續三周。結果顯示過繼輸入的來源於人EGFR重組DNA疫苗hEe-p免疫小鼠的Ig具有顯著的腫瘤抑制作用。作為對照，把純化的Ig與固定的EGFR陽性腫瘤細胞或EGFR陰性腫瘤細胞在4℃下振蕩1小時以吸附Ig，共重複4次。結果發現來源於人EGFR重組DNA疫苗hEe-p免疫小鼠的Ig的抗腫瘤效應由於被EGFR陽性腫瘤細胞(LL/2c，MA782/5S)預吸附而消除，但對EGFR陰性腫瘤細胞(H22)仍然有效(圖11C)。
除上述過表達EGFR的肺癌、乳腺癌外，EGFR分子疫苗對其他各種過表達EGFR的實體瘤，包括卵巢癌、結腸癌、前列腺癌、胃癌、膀胱癌、頭頸部鱗癌和膠質瘤等，也有較好的抗腫瘤作用效應。
七、EGFR分子疫苗的抗腫瘤作用原理及生物安全性現代免疫學認為，抗原或抗原表位與相應受體間的相互識別除特異性外，還存在一定程度的伸展性(plasticity)、是混雜性(promiscuity)或退變性(degeneracy)，從而通過分子模擬(molecular mimicry)機制誘導針對自身抗原的免疫交叉反應，打破免疫耐受。
本發明發現EGFR分子作為一種免疫交叉抗原，能打破機體對自身EGFR分子的免疫耐受，誘導機體產生針對EGFR分子自身免疫樣交叉反應，這些免疫反應包括針對EGFR分子的主動免疫反應(包括細胞免疫反應和體液免疫反應)和被動免疫反應(過繼免疫反應)。
為了研究EGFR分子疫苗的抗腫瘤作用原理，我們首先對EGFR分子疫苗免疫小鼠進行體液免疫檢測，包括應用流式細胞術、Western印跡、免疫沉澱等驗證小鼠/兔免疫血清中抗EGFR自身抗體的存在，應用ELISA檢測小鼠/兔免疫血清中抗EGFR抗體的滴度及抗體類型，應用免疫組織化學法檢測免疫小鼠腫瘤組織內的自身抗體，應用腫瘤細胞成集落實驗、血清過繼免疫實驗檢測自身抗體的功能等。
每隻小鼠在免疫前及免疫後每周經鼠尾靜脈採血或處死小鼠時取血，收集血清備用。Western Blot檢測表明重組EGFR分子疫苗免疫誘導的抗體可以特異識別相應的免疫原(重組EGFR蛋白或表達於腫瘤細胞上的EGFR)，但不能識別EGFR陰性細胞。同時，用流式細胞儀對所獲得的抗體空間表位進行識別，也發現重組EGFR分子疫苗能誘導產生特異的抗體，可以識別表達於腫瘤細胞表面的EGFR。ELISA自身抗體測定結果顯示，在初次免疫2周後重組EGFR分子疫苗免疫小鼠開始產生抗小鼠自身EGFR抗體，滴度可達1∶100至1∶5000，以後逐漸升高，至第4周抗體滴度可達1∶10000至1∶500000，到第8周抗體滴度仍能維持在1∶500至1∶1000，而相應的對照組則檢測不到明顯的抗體產生。對所產生的抗體亞型作進一步檢測，發現EGFR分子疫苗產生的抗體以IgG為主。圖12顯示了EGFR分子疫苗(重組蛋白疫苗和重組DNA疫苗)免疫小鼠所誘導產生的抗體類型，從圖中可以看出，重組EGFR分子疫苗明顯誘導產生了IgG1、IgG2a和IgG2b等抗體，但IgM和IgA並沒有升高，並且這些抗體可被CD4抗體阻斷，但不能被抗CD8、抗NK或對照抗體所阻斷，而其他對照組蛋白疫苗、佐劑和生理鹽水組則不能檢測到特異抗體。
EGFR分子疫苗還誘導了細胞免疫。對EGFR分子疫苗免疫的小鼠進行的細胞免疫檢測主要包括應用51Cr釋放法測定CTL活性，應用ELISPOT檢測細胞因子水平(主要是免疫血清中IFN-γ和IL-4濃度)，應用免疫細胞去除(Depletion ofimmune cell subsets)實驗檢測T細胞類型(CD4+T淋巴細胞、CD8+T淋巴細胞或NK細胞等)。
重組chEGFR疫苗免疫小鼠後，取脾單個核細胞作ELISPOT檢測，發現脾單個核細胞中存在大量抗原特異B細胞，而經重組mEGFR疫苗免疫後也能發現少量特異B細胞的存在，但空白組及佐劑或空載體組未能發現有統計意義的特異B細胞。重組EGFR分子疫苗免疫小鼠3周後，取免疫小鼠脾T細胞，並用健康脾單個核細胞為抗原呈遞細胞加以免疫原在體外再刺激擴增可能已活化T細胞。結果顯示經重組chEGFR分子疫苗免疫後均含有較多量的IFN-及IL-4產生細胞，而重組mEGFR分子疫苗在體外再刺激源於chEGFR疫苗免疫小鼠的脾T細胞也含有較多的IFN-及IL-4產生細胞，明顯多於同等條件下未體外刺激的T細胞。進一步以標準的51Cr釋放法測定了重組EGFR分子疫苗免疫後脾T細胞對鼠、人等相關腫瘤細胞的特異殺傷活性。結果顯示重組chEGFR分子疫苗免疫後的小鼠脾T細胞在40∶1時對EGFR陽性表達LL/2c、MA781/5S腫瘤細胞的特異殺傷活性分別達40.27％、42.83％，但對重組mEGFR分子疫苗免疫的小鼠脾T細胞對MA781/5S或LLC腫瘤細胞、以及EGFR陰性C26細胞未發現殺傷活性，同時這種作用可被相應抗CD8及MHC-I單克隆抗體阻斷，而不能被抗CD4及MHC-II單克隆抗體阻斷，其他對照組脾T細胞則未檢測到有統計意義的殺傷活性。圖13顯示了EGFR重組DNA疫苗免疫小鼠所表現出的CTL效應，從圖中可以看出人EGFR重組DNA疫苗hEe-p免疫小鼠來源的T細胞比其他對照組來源的T細胞對EGFR陽性的腫瘤細胞有著更高的細胞毒性，並且這種細胞毒性在體外可被抗CD8或抗MHC I單抗所阻斷，但不能被抗CD4單抗所阻斷，顯示這種細胞殺傷活性來源於MHC I依賴的CD8+T細胞。另外，激活的脾細胞對YAC-1靶細胞沒有顯示出NK活性的增加。此外，過繼輸注人EGFR重組DNA疫苗hEe-p免疫小鼠來源的CD4剔除(CD8+)或CD8剔除(CD4+)T細胞顯示出對EGFR陽性腫瘤細胞的抗腫瘤作用，但對同源的EGFR陰性腫瘤細胞的抗腫瘤作用不明顯，而相應的對照組也未顯示出抗腫瘤作用效果。
本發明還對EGFR分子疫苗免疫小鼠的潛在長期毒性進行了觀察。研究中沒有觀察到諸如小鼠體重減輕、皮毛皺縮、食慾減退、壽命縮短等明顯的毒副作用。通過對免疫小鼠的肝、肺、脾、腦等器官的顯微病理檢查，沒有發現病理變化，並且免疫螢光染色也未在主要臟器中發現自身抗體沉著(圖14)。
圖1 同源分子疫苗的作用原理示意2 重組EGFR質粒圖譜A.重組EGFR真核表達質粒圖譜；B.重組EGFR原核表達質粒圖譜；C.重組EGFR酵母表達質粒圖譜；D.EGFR重組腺病毒穿梭質粒圖譜；E.EGFR重組Lentivirus病毒前體質粒圖譜圖3 EGFR重組蛋白疫苗的構建流程示意4 EGFR重組病毒疫苗的構建流程示意圖A.EGFR重組腺病毒疫苗的構建流程示意圖；B.EGFR重組Lentivirus病毒疫苗的構建流程示意5 RGD修飾的腺病毒重組EGFR疫苗構建流程示意6 納米靶向EGFR分子疫苗抗腫瘤作用原理示意7.重組EGFR DNA疫苗的抗腫瘤保護性免疫效應A.LL/2c Lewis肺癌荷瘤的免疫小鼠的腫瘤體積變化；B.MA782/5S乳腺癌荷瘤的免疫小鼠的腫瘤體積變化；C.LL/2c Lewis肺癌荷瘤的免疫小鼠的生存率；D.MA782/5S乳腺癌荷瘤的免疫小鼠的生存率。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空質粒對照；Saline，生理鹽水對照。
圖8.重組EGFR DNA疫苗的抗腫瘤治療性免疫效應A.LL/2c Lewis肺癌荷瘤的免疫小鼠的腫瘤體積變化；B.MA782/5S乳腺癌荷瘤的免疫小鼠的腫瘤體積變化；C.LL/2c Lewis肺癌荷瘤的免疫小鼠的生存率；D.MA782/5S乳腺癌荷瘤的免疫小鼠的生存率。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空質粒對照。
圖9 重組EGFR蛋白疫苗的抗腫瘤免疫效應A.保護性免疫效應；B.治療性免疫效應；C.荷瘤(MA782/5S乳腺癌)小鼠生存曲線。edCER，雞EGFR胞外段重組蛋白疫苗；edMER，鼠EGFR胞外段重組蛋白疫苗；Adj，鋁佐劑；NS，生理鹽水。
圖10 重組EGFR蛋白疫苗的抗腫瘤轉移效應，A.LL/2c腫瘤肺部轉移灶數量；B.LL/2c腫瘤轉移後肺溼重。edCER，雞EGFR胞外段重組蛋白疫苗；edMER，鼠EGFR胞外段重組蛋白疫苗；Adj，鋁佐劑；NS，生理鹽水。
圖11 EGFR分子疫苗的體外(in vitro)抑制腫瘤細胞生長效應及體內(invivo)過繼免疫抗腫瘤效應A.EGFR重組DNA疫苗hEe-p免疫小鼠來源的Ig對EGFR陽性腫瘤細胞(A549，LL/2c，MA782/5S)和EGFR陰性腫瘤細胞(H22 andMMT-06052)的生長抑制狀況。B.用未免疫的正常小鼠來源的Ig對腫瘤細胞的生長抑制狀況。C.EGFR重組DNA疫苗hEe-p免疫小鼠來源的Ig的體內(in vivo)過繼免疫抗腫瘤效應。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空質粒對照；Saline，生理鹽水對照。
圖12 EGFR分子疫苗誘導產生的抗體類型A.EGFR重組DNA疫苗誘導產生的抗體類型；B.EGFR重組蛋白疫苗誘導產生的抗體類型。hEe-p，人EGFR胞外段DNA疫苗；mEe-p，鼠EGFR胞外段DNA疫苗；c-p，空質粒對照；Saline，生理鹽水對照；edCER，雞EGFR胞外段重組蛋白疫苗；edMER，鼠EGFR胞外段重組蛋白疫苗；Adj，鋁佐劑；NS，生理鹽水。
圖13 EGFR重組DNA疫苗免疫小鼠的CTL效應A.人EGFR重組DNA疫苗hEe-p及其對照組免疫小鼠來源的T細胞對LL/2c Lewis肺癌細胞在不同E∶T比例下的51Cr釋放分析結果；B.過繼輸注2×107個來源於人EGFR重組DNA疫苗hEe-p免疫小鼠的CD4剔除(CD8+)T細胞對EGFR陽性的LL/2c Lewis肺癌細胞和EGFR陰性的B16黑色素瘤細胞的抗腫瘤作用；C.過繼輸注2×107個來源於人EGFR重組DNA疫苗hEe-p免疫小鼠的CD8剔除(CD4+)T細胞對EGFR陽性的LL/2c Lewis肺癌細胞和EGFR陰性的B16黑色素瘤細胞的的抗腫瘤作用；D.過繼輸注2×107個來源於人EGFR重組DNA疫苗hEe-p免疫小鼠的CD4剔除(CD8+)T細胞對EGFR陽性的MA782/5S乳腺癌細胞和EGFR陰性的MethA纖維肉瘤細胞的的抗腫瘤作用；E.過繼輸注2×107個來源於人EGFR重組DNA疫苗hEe-p免疫小鼠的CD8剔除(CD4+)T細胞對EGFR陽性的MA782/5S乳腺癌細胞和EGFR陰性的MethA纖維肉瘤細胞的抗腫瘤作用。 人EGFR重組DNA疫苗hEe-p免疫小鼠來源的T細胞過繼免疫的荷瘤小鼠； 鼠EGFR重組DNA疫苗mEe-p免疫小鼠來源的T細胞過繼免疫的荷瘤小鼠； 非免疫小鼠來源的T細胞過繼免疫的荷瘤小鼠。
圖14腫瘤細胞及組織自身抗體的免疫螢光顯微圖譜A.EGFR分子疫苗免疫的LL/2c Lewis肺癌細胞的自身抗體沉著；B.EGFR分子疫苗免疫的MA782/5S鼠乳腺癌細胞的自身抗體沉著；C.未免疫的LL/2c Lewis肺癌細胞無自身抗體沉著；D.未免疫的MA782/5S鼠乳腺癌細胞無自身抗體沉著；E.CD4+T阻斷的EGFR分子疫苗免疫的LL/2c Lewis肺癌細胞無自身抗體沉著；F.CD4+T阻斷的EGFR分子疫苗免疫的MA782/5S鼠乳腺癌細胞無自身抗體沉著；G CD8+T阻斷的EGFR分子疫苗免疫的LL/2c Lewis肺癌細胞顯示自身抗體沉著；H.CD8+T阻斷的EGFR分子疫苗免疫的MA782/5S鼠乳腺癌細胞顯示自身抗體沉著；I.EGFR分子疫苗免疫小鼠的肝臟無自身抗體沉著；J.EGFR分子疫苗免疫小鼠的腎臟無自身抗體沉著；K.未免疫小鼠的肝臟無自身抗體沉著；L.未免疫小鼠的腎臟無自身抗體沉著。
具體實施例方式
實施例1 EGFR重組DNA疫苗依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GACCATGGAGGAAAAGAAAGTTTGC 3』，5』ACGAATTCTTAGGACGGGATCTTAGGCCCA3』；小鼠的引物為5』GACCATGGAGGAAAAGAAAGTCTGC 3』，5』ACGAATTCTTAATAGATGGTATCTTTGGC 3』；雞的引物為5』GACCATGGAGGAGAAGAAAGTTTGTC 3』，5』ACGAATTCTTAAGATGGAGTTTTGGAGCC 3』)，分別以人肺癌細胞株A431、小鼠肺癌細胞株LL2和雞胚的總RNA為模板進行RT-PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用NcoI和EcoRI酶切，收集1.9kb片段並純化，插入到用NcoI和EcoRI雙酶切的pORF-MCS(InvivoGen公司)載體，篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為pORF-hEGFR、pORF-mEGFR和pORF-chEGFR。
同樣地，設計針對於真核表達載體pcDNA3.1(+)(Invitrogen公司)的PCR引物，分別是人的引物5』GAGCTAGCATGGAGGAAA AGAAAGTTTGC 3』，5』CACTCGAGTTAGGACGGGATCTTAGGCCCA 3』；小鼠的引物為5』GAGCTAGCATGGAGGAAAAGAAAGTCTGC 3』，5』CACTCGAG TTAATAGATGGTATCTTTGGC 3』；雞的引物為5』GAGCTAGCATGGAGGAGAA GAAAGTTTGTC 3』，5』CACTCG AGTTAAGATGGAGTTTTGGAGCC 3』，依前述RNA模板進行RT-PCR擴增。將純化的1.9kb的PCR產物用NheI和XhoI雙酶切，插入到對應的雙酶切載體pcDNA3.1(+)中，篩選重組質粒，將重組質粒分別命名為pcDNA-hEGFR、pcDNA-mEGFR和pcDNA-chEGFR。
這些真核表達質粒pORF-EGFR或pcDNA-EGFR等轉染入CHO、NIH3T3、Vero等細胞系，觀察並用SDS-PAGE、ELISA、Western Blot等技術檢測其表達EGFR的情況和水平。用鹼法抽提經過鑑定的重組EGFR分子表達質粒，用超離心、超濾等方法去除大腸桿菌內毒素，即得到純淨的質重組粒DNA，這些質粒DNA即可作為DNA疫苗進行免疫。
概括地講，EGFR重組DNA疫苗的構建過程是依據GenBank等各公開的資料庫所收藏的各種生物來源(如人、小鼠、大鼠、雞、鮐、果蠅等，見SEQ IDNO 1-14)的關於EGFR分子的序列(包括基因、cDNA、mRNA和胺基酸序列)用常規方法設計引物或探針，用PCR、RT-PCR、雜交等技術，從各種商品化的基因文庫、cDNA文庫(ClonTech，Strategene公司等生產的各種基因文庫、cDNA文庫)或各種細胞系(如人肺癌細胞株A431、鼠Lewis肺癌細胞株LL2等)、組織(如肺癌、乳腺癌組織、果蠅、雞胚等)等中克隆分離出不同種屬生物來源的EGFR分子的胞外區段的cDNA，或進一步用基因直接進化技術篩選到的具有強免疫原性的EGFR分子。各種來源的EGFR cDNA的胞外段序列經測序鑑定後，用分子克隆技術插入真核生物表達質粒(如pcDNA3.1、pORF-mcs、pBLAST-mcs、pSecTag2等)，篩選重組表達質粒，經限制性酶切分析鑑定後轉染入CHO、NIH3T3、Vero等細胞系，觀察並用SDS-PAGE、ELISA、Western Blot等技術檢測其表達EGFR的情況和水平。用鹼法抽提經過鑑定的重組EGFR分子表達質粒，用超離心、超濾等方法去除大腸桿菌內毒素，即得到純淨的質重組粒DNA，這些質粒DNA即可作為DNA疫苗進行免疫。
選擇6-8周齡小鼠，用常規方法建立各種荷瘤小鼠模型，每隻小鼠注射100gEGFR重組表達質粒，每周1次，連續4周。觀察荷瘤小鼠的生長情況及腫瘤的發展情況，8周後處死小鼠，收集免疫小鼠血清及各種臟器，用流式細胞術、ELISA、Western blot等方法檢測體液免疫反應，用Cr51、ELISpot等檢測細胞免疫反應，用免疫組化方法檢測免疫的毒、副作用。進一步提純免疫小鼠血清，按常規方法建立裸鼠的腫瘤模型，進行過繼免疫治療，觀察腫瘤的生長情況。
實施例2 EGFR重組蛋白疫苗(E.coli表達)同前文所述，依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GACCATGGAGGAAAAGAAAGTTTGC 3』，5』ACAGATCTAGGACGGGATCTTAGGCCCA 3』；小鼠的引物為5』GACCATGGAGGAAAAGAAAGTCTGC 3』，5』ACAGATCTATAGATGGTATCTTTGGC 3』；雞的引物為5』GACCATGGA GGAGAAGAAAGTTTGTC 3』，5』ACAGATCTAGATGGAGTTTTGGAGCC 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用NcoI和BglII酶切，收集1.9kb片段並純化，插入到用NcoI和BglII雙酶切的pQE60(QIAGEN公司)載體，篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為pQE-hEGFR、pQE-mEGFR和pQE-chEGFR。
同樣地，可針對其他不同的原核表達載體(如pET32、pLLp、pSE420等)設計PCR引物，以構建其他的重組原核表達質粒。
經限制性酶切分析等鑑定後的各重組原核表達質粒，轉化不同的大腸桿菌宿主(如E.coli TOP10F′、E.coli BL21(DE3)pLys、E.coli M15、E.coli DH5、E.coliJM109等)，觀察並用SDS-PAGE、ELISA、Western Blot等技術檢測其表達EGFR的情況和水平，確定最適的大腸桿菌表達宿主菌株。用經確證的重組EGFR表達質粒轉化最適大腸桿菌菌株，建立穩定表達菌株和各級種子庫。重組菌株大量搖瓶培養或發酵，低溫離心收集菌體，菌體經PBS重懸後用超聲法破碎細胞，用離子交換層析、親和層析等方法分離純化重組EGFR蛋白，此重組的EGFR蛋白質即可作為蛋白疫苗進行免疫。
選擇6-8周齡小鼠，用常規方法建立各種荷瘤小鼠模型，每隻小鼠注射5-50gEGFR重組蛋白，每周1次，連續4周。觀察荷瘤小鼠的生長情況及腫瘤的發展情況，8周後處死小鼠，收集免疫小鼠血清及各種臟器，用流式細胞術、ELISA、Western blot等方法檢測體液免疫反應，用Cr51、ELISpot等檢測細胞免疫反應，用免疫組化方法檢測免疫的毒、副作用。進一步提純免疫小鼠血清，按常規方法建立裸鼠的腫瘤模型，進行過繼免疫治療，觀察腫瘤的生長情況。
實施例3 EGFR重組蛋白疫苗(酵母Pichia pastoris表達)
同前文所述，依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』ATACTCGAGAAAAGAGAGCTGGAGGAAAAGAAAG 3』，5』GCTCTAGAATGGCACAGGTGGCACA 3』；小鼠的引物為5』ATGCTCGAGAAAA GAGAGTTGGAGGAAAAGAAAGTC 3』，5』AAGCGGCCGCCATAGATGGTATCTTTG 3』；雞的引物為5』ATACTCGAGAAAAGAGAGGTGGAGGAGAAGAAAG 3』，5』CGTCTAGAAGATGGAGTTTTGGAG 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用XhoI和XbaI(對小鼠的克隆為XhoI和NotI)酶切，收集1.9kb片段並純化，插入到用XhoI和XbaI雙酶切(對小鼠的克隆為XhoI和NotI雙酶切)的pPICZ A(Invitrogen公司)載體，轉化E.coli篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為酵母表達質粒pYE-hEGFR、pYE-mEGFR和pYE-chEGFR。這些酵母表達質粒經PmeI酶切線性化後，用電穿孔法轉化酵母菌株X33或GS115，用Zeocin抗性篩選穩定轉化子，在MMH(Minimal Methanol with histidine，MMH)和MDH(Minimal Dextrose with histidine，MDH)瓊脂平板上鑑定和挑選Mut+轉化子。挑選6-10個Mut+轉化子進行小規模表達，用SDS-PAGE、Western Blot、ELISA等方法對所表達的重組蛋白進行鑑定。選擇其中表達效率最高的Mut+轉化子做大規模表達，建立各級酵母表達種子庫。重組菌株大量搖瓶培養或發酵，低溫離心收集菌體，菌體經PBS重懸後用超聲法破碎細胞，用離子交換層析、親和層析等方法分離純化重組EGFR蛋白，此重組的EGFR蛋白質即可作為蛋白疫苗進行免疫。
同樣地，也可用其他的酵母表達系統構建類似的酵母重組表達質粒。
選擇6-8周齡小鼠，用常規方法建立各種荷瘤小鼠模型，每隻小鼠注射5-50gEGFR重組蛋白，每周1次，連續4周。觀察荷瘤小鼠的生長情況及腫瘤的發展情況，8周後處死小鼠，收集免疫小鼠血清及各種臟器，用流式細胞術、ELISA、Western blot等方法檢測體液免疫反應，用Cr51、ELISpot等檢測細胞免疫反應，用免疫組化方法檢測免疫的毒、副作用。進一步提純免疫小鼠血清，按常規方法建立裸鼠的腫瘤模型，進行過繼免疫治療，觀察腫瘤的生長情況。
實施例4 EGFR重組腺病毒疫苗同前文所述，依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GAAGATCTATGGAGGAAAAGAAAGTTTGC 3』，5』ACGATATCTTAAGGACGGGATCTTAGGCCCA 3』；小鼠的引物為5』GAAGATCTATGGAGGAAAAGAAAGTCTGC 3』，5』ACGATATCTTAATAGATGGTATCTTTGGC 3』；雞的引物為5』GAAGATCTATGGAGGAGAAGAAAGTTTGTC 3』，5』ACGATATCTTAAGATGGAGTTTTGGAGCC 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用BglII和EcoRV酶切，收集1.9kb片段並純化，插入到用BglII和EcoRV雙酶切的pShuttle-CMV(Quantum Biotechnologies公司)載體，篩選重組質粒。候選的重組質粒經限制性酶切分析及PCR擴增等雙重鑑定，命名為腺病毒穿梭表達質粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR(圖2D)。分別將經PmeI酶切的腺病毒穿梭表達載體pShuttle-EGFR與包含腺病毒基因組的骨架載體pAdEasy-1或pAdEasy-2共轉化E.coli BJ5183，得到重組腺病毒載體質粒pAd-hEGFR、pAd-mEGFR和pAd-chEGFR。這些重組腺病毒載體質粒經PacI酶切後，用磷酸鈣-DNA共沉澱法轉染入腺病毒包裝細胞株293細胞中，得到相應的重組腺病毒Ad-hEGFR、Ad-mEGFR和Ad-chEGFR。用PCR、Western blot和酶切分析確證EGFR基因構建入重組腺病毒載體中並能在真核細胞中得到正確有效的表達，用超離心法大量製備重組腺病毒Ad-EGFR，用上層瓊脂法、TCID50等法測定每批次的重組腺病毒的滴度(pfu)。利用293細胞大量擴增經確證的EGFR重組腺病毒疫苗，用超離心、超濾等技術分離、純化重組腺病毒，此經純化的EGFR重組腺病毒即可作為疫苗進行免疫。
同前文所述，選擇6-8周齡小鼠，用常規方法建立各種荷瘤小鼠模型，每隻小鼠注射1×109 PFU的EGFR重組腺病毒，每周1次，連續4周。觀察荷瘤小鼠的生長情況及腫瘤的發展情況，8周後處死小鼠，收集免疫小鼠血清及各種臟器，用流式細胞術、ELISA、Western blot等方法檢測體液免疫反應，用Cr51、ELISpot等檢測細胞免疫反應，用免疫組化方法檢測免疫的毒、副作用。進一步提純免疫小鼠血清，按常規方法建立裸鼠的腫瘤模型，進行過繼免疫治療，觀察腫瘤的生長情況。
實施例5 EGFR重組Lentivirus病毒疫苗如前文所述，本發明利用ViraPowerTMLentiviral GatewayExpression Kit(Invitrogen公司)構建EGFR重組Lentivirus病毒，其具體構建過程是依據GenBank等各公開的資料庫所收藏的人、小鼠、雞、的EGFR分子的cDNA序列(分別對應於SEQ ID NO 1-5，7-9，19)設計PCR引物(人的引物為5』GACCATGGAGGAAAAGAAAGTTTGC 3』，5』ACGATATCAGGACGGGA TCTTAGGCCCA 3』；小鼠的引物為5』GACCATGGAGGAAAAGAAAGTCTGC 3』，5』ACGATATCATAGATGGTATCTTTGGC 3』；雞的引物為5』GACCATGGAGGAGAAGAAAGTTTGTC 3』，5』ACGATATCAGATGGAGTTTTGGAGCC 3』)，分別以pORF-hEGFR、pORF-mEGFR和pORF-chEGFR等為模板進行PCR擴增，電泳收集純化擴增的EGFR片段(均為1.9kb)，然後進行PCR產物亞克隆。把PCR亞克隆測序確證後，用NcoI和EcoRV酶切，收集1.9kb片段並純化，插入到用NcoI和EcoRV雙酶切的pENTR11(Invitrogen公司)載體中，篩選重組質粒，命名為pENTR-hEGFR、pENTR-mEGFR和pENTR-chEGFR(圖2E)。分別將這些載體pENTR-EGFR與包含Lentivirus病毒基因組的骨架載體pLenti6/V5-DEST共轉化E.coli DH5，得到重組Lentivirus病毒載體質粒pLenti-hEGFR、pLenti-mEGFR和pLenti-chEGFR(圖2E)。這些重組Lentivirus病毒載體質粒與包裝混合物一起(ViraPowerTMPackagingMix)用磷酸鈣-DNA共沉澱法轉染入Lentivirus病毒包裝細胞株293FT細胞中，得到相應的重組Lentivirus病毒Lenti-hEGFR、Lenti-mEGFR和Lenti-chEGFR。用PCR、Western blot等確證EGFR基因構建入重組Lentivirus病毒載體中並能在真核細胞中得到正確有效的表達。利用293FT細胞大量擴增經確證的EGFR重組Lentivirus病毒疫苗，用超離心、超濾等技術分離、純化重組Lentivirus病毒，此經純化的EGFR重組Lentivirus病毒即可作為疫苗進行免疫。
實施例6 甘露糖化腺病毒重組EGFR疫苗如前文所述，用常規方法擴增EGFR重組腺病毒(I代、II代均可)，層析或超離心法純化重組腺病毒。將70mg mannan(sigma)溶於5ml 0.1M的磷酸鹽緩衝液(pH6.0)中，終濃度14mg/ml，加45ml 0.01M高碘酸鈉溶液，在4℃下混合氧化60分鐘，加入10l乙二醇，在4℃下孵育30分鐘，即得Ox-M(Oxidative Mannan)混合物。將Ox-M混合物倒入用重碳酸鹽緩衝液(pH6.0-9.0)平衡Sephadex-G25層析柱進行層析分離，OX-M即被洗入2ml的空容器。將純化的Ox-M與1×1014腺病毒顆粒混合，室溫過夜，即獲得所需Ox-M-腺病毒。在Ox-M-腺病毒中加入1mg/ml硼氫化鈉，室溫放置3小時即得Red-M-腺病毒。Ox-M-腺病毒與Red-M-腺病毒經超濾脫鹽、濃縮後，過濾細菌，小管分裝，-80℃低溫保存。此經純化的甘露糖化重組EGFR腺病毒即可作為疫苗進行免疫。
同前文所述，選擇6-8周齡小鼠，用常規方法建立各種荷瘤小鼠模型，每隻小鼠注射1×1010PFU的重組EGFR甘露糖化腺病毒疫苗，每周1次，連續4周。觀察荷瘤小鼠的生長情況及腫瘤的發展情況，8周後處死小鼠，收集免疫小鼠血清及各種臟器，用流式細胞術、ELISA、Western blot等方法檢測體液免疫反應，用Cr51、ELISpot等檢測細胞免疫反應，用免疫組化方法檢測免疫的毒、副作用。進一步提純免疫小鼠血清，按常規方法建立裸鼠的腫瘤模型，進行過繼免疫治療，觀察腫瘤的生長情況。
實施例7 RGD修飾的腺病毒重組EGFR疫苗的構建如前文所述，本發明利用AdEasy系統構建RGD修飾腺病毒的重組EGFR疫苗，其具體過程是腺病毒基因組骨架質粒pAdEasy-1和pAdEasy-2經限制性內切酶SpeI(Sp)酶切後，用T4 DNA聚合酶補平(filling，f)末端，再用PacI(P)酶切，分別電泳回收6211bp和3579bp的片段，分別命名為AdFiber I/Sp/f/P和AdFiber II/Sp/f/P，該片段包含了完整的腺病毒纖毛(Adenovirus fiber)基因。把AdFiber I/Sp/f/P和AdFiber II/Sp/f/P片段插入經BamHI酶切-T4 DNA聚合酶補平-PacI酶切處理(BamHI/filling/PacI-digested)的pShuttle載體，將所得的重組質粒分別命名為pSh-AdFiber I和pSh-AdFiber II。pSh-AdFiber I用NheI酶切-T4DNA聚合酶補平-KpnI酶切處理(NheI/filling/KpnI)，電泳回收2090bp的片段AdFiber I/Nh/f/K，將該片段插入到經SmaI/KpnI雙酶切的pUC18載體中，所得的重組質粒命名為pUC-AdFiber I；而pSh-AdFiber II用AvrII酶切-T4 DNA聚合酶補平-HindIII酶切處理(AvrII/filling/HindIII)，電泳回收838bp的片段AdFiberI/A/f/H，將該片段插入到經SmaI/HindIII雙酶切的pUC18載體中，所得的重組質粒命名為pUC-AdFiber II。設計一系列PCR引物以便以pUC-AdFiber I和pUC-AdFiberII為模板擴增腺病毒疣足(Adenovirus knob，Ad-knob)基因序列，引物分別是F1(5』GAAAGCTAGC CCTGCAAACATCA 3』)、R1(5』ACTCCCGGGAGTTGTGTCTCCTGTTTCCTG 3』)、F2(5』ACTCCCGGGAGTGCATACTCTATGTCA 3』)、R2(5』TATGGTAC CGGGAGGTGGTGA 3』)、F3(5』AACCTAGGGAGGTTAACCTAAGCACTG 3』)、和R3(5』CTCAAGCTTTTTGGAATTGTTTGA 3』)。以引物F1-R1、F2-R2、F3-R1和F2-R3分別進行第一輪PCR，得到產物PCR1、PCR2、PCR3和PCR4，再以F1-R2和F3-R3為引物，以第一次擴增產物PCR1與PCR2、PCR3與PCR4為模板進行第二輪PCR擴增，得到PCR產物PCR1-PCR2(PCR I)、PCR3-PCR4(PCR II)，將第二輪PCR擴增的PCR I和PCR II插入到經EcoRV酶切的pBR322載體中，所得到的重組質粒命名為pBR-PCR I和pBR-PCR II。把RGD-4C雙螺旋寡聚核苷酸(RGD-4C duplex)5』TGTGACTGCCGCGGAGACTGTTTCTGC 3』3』ACACTGACGGCGCCTCTGACAAAGACG 5』插入到SmaI酶切的pBR-PCR I and pBR-PCR II載體中，將所得到的重組質粒命名為pBR-PCR/RGD I和pBR-PCR/RGD II，並對重組結構進行測序確證。用NheI/KpnI雙酶切pBR-PCR/RGD I，電泳回收PCR/RGD I片段，然後插入到NheI/KpnI雙酶切的pUC-AdFiber I載體中，所得的重組質粒命名為pUC-AdFiber-RGD I；用AvrII/HindIII雙酶切pBR-PCR/RGD II，電泳回收PCR/RGDII片段，再插入到AvrII/HindIII雙酶切的pUC-AdFiber II載體中，將所得到的重組質粒命名為pUC-AdFiber-RGD II。然後，用SpeI/PacI雙酶切pUC-AdFiber-RGDI陽pUC-AdFiber-RGD II載體，電泳回收AdFiber-RGD I、AdFiber-RGD II片段，插入到SpeI/PacI雙酶切的pAdEasy-1、pAdEasy-2載體中，所得的重組質粒分別命名為pAdEasy-RGD I、pAdEasy-RGD II。將經PmeI線性化的腺病毒穿梭質粒pShuttle-hEGFR、pShuttle-mEGFR和pShuttle-chEGFR分別與pAdEasy-RGD I和pAdEasy-RGD II共轉化E.coli BJ5183，所得的重組質粒命名為腺病毒質粒pAd-RGD-EGFR I和pAd-RGD-EGFR II，腺病毒質粒pAd-RGD-EGFR I轉染293細胞，所得的重組腺病毒命名為Ad-RGD-EGFR I，腺病毒質粒pAd-RGD-EGFR II轉染293E4pIX細胞，所得的重組腺病毒命名為Ad-RGD-EGFR II。
經純化的Ad-RGD-EGFR I和Ad-RGD-EGFR II可作為疫苗進行免疫，對腫瘤血管內皮細胞具有特異的靶向性。
實施例8 重組EGFR分子疫苗的藥效學觀察藥效學研究是觀察和驗證重組EGFR分子疫苗的抗腫瘤作用的重要指標。在本發明中，藥效學觀察的實驗包括普通荷瘤小鼠(含保護性免疫實驗、治療性免疫實驗、腫瘤轉移模型實驗)、兔免疫試驗、猴免疫試驗、裸鼠過繼免疫實驗、劑量依存實驗、體外細胞實驗等。這裡主要敘述以肺癌為模型的普通荷瘤小鼠實驗，其它可類比。
1.荷瘤小鼠保護性免疫實驗 隨機分組的6-8周齡C57BL/6小鼠雙側後肢股四頭肌肌肉交叉注射EGFR分子疫苗，每周一次，連續四周。第5周各組小鼠均右腋皮下接種LL/2腫瘤細胞1×106。分別在首次免疫第0、1、3、5周經鼠尾靜脈或處死小鼠時採血。離心後(5000轉/分，3分鐘)收集血清，-20℃保存備用。
2.荷瘤小鼠治療性免疫實驗 隨機分組的6-8周齡C57BL/6小鼠均右腋皮下接種LL/2腫瘤細胞1×106。接種後第4天(可觸及腫瘤)，隨機分組，小鼠後肢肌肉交叉注射。每周一次，連續四周。各組分別在首次免疫第0、2、4、6周經鼠尾靜脈或處死小鼠時採血。離心後(5000轉/分，3分鐘)收集血清，-20℃保存備用。觀察並記錄腫瘤重量、體積及生存曲線。
3.荷瘤小鼠轉移模型實驗 選6-8周齡C57BL/6雌性小鼠，隨機分組，小鼠後肢肌肉交叉注射疫苗及對照組。每周一次，連續四周。2周後，取對數期生長的LL/2細胞，按2×105/只接種於C57BL/6小鼠右後腿肌肉內，繼續上述疫苗注射，4周後斷頸處死小鼠，取肺稱重，檢查並計數肺部轉移灶後，用10％中性緩衝福馬林固定。
序列表110深圳市清華源興生物醫藥科技有限公司120疫苗製備方法和抗腫瘤疫苗130CGCNS31755150CN02159602.61512002-12-2716027170PatentIn version 3.121012113633212DNA213人(homo sapiens)220
221CDS222(1)..(3630)223
4001atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560
ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235 240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320
gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550 555560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575
gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615 620acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat ggg 1920Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640cct aag atc ccg tcc atc gcc act ggg atg gtg ggg gcc ctc ctc ttg 1968Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu645 650 655ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg cga agg cgc cac 2016Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His660 665 670atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag gag agg gag ctt 2064Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu675 680685gtg gag cct ctt aca ccc agt gga gaa gct ccc aac caa gct ctc ttg 2112Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu690695 700agg atc ttg aag gaa act gaa ttc aaa aag atc aaa gtg ctg ggc tcc 2160Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser705 710715 720ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc cca gaa ggt gag 2208Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu725 730735aaa gtt aaa att ccc gtc gct atc aag gaa tta aga gaa gca aca tct 2256Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser740 745 750ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac gtg atg gcc agc 2304Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser755760765gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc tgc ctc acc tcc 2352Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser770775 780acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc tgc ctc ctg gac 2400Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp785 790795 800tat gtc cgg gaa cac aaa gac aat att ggc tcc cag tac ctg ctc aac 2448Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn805 810815tgg tgt gtg cag atc gca aag ggc atg aac tac ttg gag gac cgt cgc 2496Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg820 825 830
ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg gtg aaa aca ccg 2544Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro835 840845cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctg ggt gcg 2592Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala850 855860gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg cct atc aag tgg 2640Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp865 870 875 880atg gca ttg gaa tca att tta cac aga atc tat acc cac cag agt gat 2688Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp885 890 895gtc tgg agc tac ggg gtg acc gtt tgg gag ttg atg acc ttt gga tcc 2736Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser900 905 910aag cca tat gac gga atc cct gcc agc gag atc tcc tcc atc ctg gag 2784Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu915 920 925aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc atc gat gtc tac 2832Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr930 935 940atg atc atg gtc aag tgc tgg atg ata gac gca gat agt cgc cca aag 2880Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys945 950 955 960ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc cga gac ccc cag 2928Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln965 970975cgc tac ctt gtc att cag ggg gat gaa aga atg cat ttg cca agt cct 2976Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro980 985990aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa gaa gac atg gac 3024Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp99510001005gac gtg gtg gat gcc gac gag tac ctc atc cca cag cag ggc ttc 3069Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe101010151020ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc tct ctg 3114Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu102510301035agt gca acc agc aac aat tcc acc gtg gct tgc att gat aga aat 3159Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn104010451050ggg ctg caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga 3204Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg105510601065tac agc tca gac ccc aca ggc gcc ttg act gag gac agc ata gac 3249Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp107010751080
gac acc ttc ctc cca gtg cct gaa tac ata aac cag tcc gtt ccc 3294Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro1085 10901095aaa agg ccc gct ggc tct gtg cag aat cct gtc tat cac aat cag 3339Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln1100 11051110cct ctg aac ccc gcg ccc agc aga gac cca cac tac cag gac ccc 3384Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro1115 11201125cac agc act gca gtg ggc aac ccc gag tat ctc aac act gtc cag 3429His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln1130 11351140ccc acc tgt gtc aac agc aca ttc gac agc cct gcc cac tgg gcc 3474Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala1145 11501155cag aaa ggc agc cac caa att agc ctg gac aac cct gac tac cag 3519Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln1160 11651170cag gac ttc ttt ccc aag gaa gcc aag cca aat ggc atc ttt aag 3564Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys1175 11801185ggc tcc aca gct gaa aat gca gaa tac cta agg gtc gcg cca caa 3609Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln1190 11951200agc agt gaa ttt att gga gca tga 3633Ser Ser Glu Phe Ile Gly Ala1205 121021022111210212PRT213人(homo sapiens)4002Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305310 315320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu645 650 655Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His660 665 670Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu675680 685Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu690695 700Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser705 710715 720Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu725 730735Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser740 745 750Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser755 760 765Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser770775780Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp785 790795800Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn805 810 815Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg820 825 830
Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro835 840845Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala850 855860Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp865870 875 880Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp885 890 895Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser900 905 910Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu915 920 925Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr930 935 940Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys945 950955960Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln965 970975Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro980 985 990Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp995 10001005Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe1010 1015 1020Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu1025 1030 1035Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn1040 1045 1050Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg1055 1060 1065Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp1070 1075 1080
Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro108510901095Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln110011051110Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro111511201125His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln113011351140Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala114511501155Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln116011651170Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys117511801185Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln119011951200Ser Ser Glu Phe Ile Gly Ala1205121021032111974212DNA213人(homo sapiens)220
221CDS222(1)..(1971)223
4003atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe354045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500505 510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515 520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610615 620acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat gga 1920Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640agc tac ata gtg tct cac ttt cca aga tca ttc tac aag atg tca gtg 1968Ser Tyr Ile Val Ser His Phe Pro Arg Ser Phe Tyr Lys Met Ser Val645 650 655cac tga 1974His2104211657212PRT213人(homo sapiens)4004Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515 520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625630 635640Ser Tyr Ile Val Ser His Phe Pro Arg Ser Phe Tyr Lys Met Ser Val645 650 655His21052112118212DNA213人(homo sapiens)220
221CDS222(1)..(2115)223
4005atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80
acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195 200205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265 270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330335
tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500505510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590
aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615620acc tac ggg cca gga aat gag agt ctc aaa gcc atg tta ttc tgc ctt 1920Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu625 630635640ttt aaa cta tca tcc tgt aat caa agt aat gat ggc agc gtg tcc cac 1968Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His645650655cag agc ggg agc cca gct gct cag gag tca tgc tta gga tgg atc cct 2016Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro660 665670tct ctt ctg ccg tca gag ttt cag ctg ggt tgg ggt gga tgc agc cac 2064Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His675680685ctc cat gcc tgg cct tct gca tct gtg atc atc acg gcc tcc tcc tgc 2112Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys690 695 700cac tga 2118His7052106211705212PRT213人(homo sapiens)4006Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp Hls Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val8590 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180185190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245 250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305310 315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610615 620Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu625630 635640Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His645650655Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro660 665670Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His675680685Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys690695 700His70521072111887212DNA213人(homo sapiens)220
221CDS222(1)..(1884)223
4007atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat 384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg 432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag 480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180185190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245 250 255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330 335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390 395400atc aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405 410415ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485 490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val SerCys Arg Asn515520525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600605aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca 8ac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615620acc tac ggg tcc taa 1887Thr Tyr Gly Ser6252108211628212PRT213人(homo sapiens)4008Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245 250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370375380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390 395400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp405 410415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450 455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu485490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn515520525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595600605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys610 615620Thr Tyr Gly Ser
62521092111218212DNA213人(homo sapiens)-220
221CDS222(1)..(1215)223
4009atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt144Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat192Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta aag240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg tac336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca aat384Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ctg432Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag480His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160
agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac atg 528Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat cca 576Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230235240aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325330 335tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360365ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375 380cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390395400atc aca ggt ttg agc tga 1218Ile Thr Gly Leu Ser405
21010211405212PRT213人(homo sapiens)40010Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150155160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met165 170175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln195200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310315320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr370 375380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Leu Ser405210112112829212DNA213人(homo sapiens)220
221CDS222(1)..(2826)223
40011atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag cgt aat tat gtg 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val20 25 30gtg aca gat cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat 144Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr3540 45gag atg gag gaa gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct 192Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro5055 60tgc cgc aaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac tca 240Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser65 70 75 80ctc tcc ata aat gct acg aat att aaa cac ttc aaa aac tgc acc tcc 288Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser85 90 95atc agt ggc gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc 336Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser100105 110ttc aca cat act cct cct ctg gat cca cag gaa ctg gat att ctg aaa 384Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys115 120125acc gta aag gaa atc aca ggg ttt ttg ctg att cag gct tgg cct gaa 432Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu130 135140aac agg acg gac ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc 480Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly145150155160agg acc aag caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac 528Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn165 170175ata aca tcc ttg gga tta cgc tcc ctc aag gag ata agt gat gga gat 576Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp180185190gtg ata att tca gga aac aaa aat ttg tgc tat gca aat aca ata aac 624Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn195200205tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata agc 672Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
210215 220aac aga ggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc 720Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala225230235 240ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc 768Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val245 250 255tct tgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac 816Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn260265270ctt ctg gag ggt gag cca agg gag ttt gtg gag aac tct gag tgc ata 864Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile275 280285cag tgc cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc aca 912Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr290295300gga cgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac ggc 960Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly305 310315 320ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac aac 1008Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn325 330335acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc 1056Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys340 345 350cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt 1104His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys355360365cca acg aat ggg cct aag atc ccg tcc atc gcc act ggg atg gtg ggg 1152Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly370375 380gcc ctc ctc ttg ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg 1200Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met385390395 400cga agg cgc cac atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag 1248Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln405 410415gag agg gag ctt gtg gag cct ctt aca ccc agt gga gaa gct ccc aac 1296Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn420 425430caa gct ctc ttg agg atc ttg aag gaa act gaa ttc aaa aag atc aaa 1344Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys435440445gtg ctg ggc tcc ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc 1392Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile450455 460cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa tta aga 1440Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg
465 470475480gaa gca aca tct ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac 1488Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr485490495gtg atg gcc agc gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc 1536Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile500 505510tgc ctc acc tcc acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc 1584Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly515 520 525tgc ctc ctg gac tat gtc cgg gaa cac aaa gac aat att ggc tcc cag 1632Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln530535 540tac ctg ctc aac tgg tgt gtg cag atc gca aag ggc atg aac tac ttg 1680Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu545550555 560gag gac cgt cgc ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg 1728Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu565570 575gtg aaa aca ccg cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa 1776Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys580 585 590ctg ctg ggt gcg gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg 1824Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val595 600605cct atc aag tgg atg gca ttg gaa tca att tta cac aga atc tat acc 1872Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr610615 620cac cag agt gat gtc tgg agc tac ggg gtg acc gtt t9g gag ttg atg 1920His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met625 630 635 640acc ttt gga tcc aag cea tat gac gga atc cct gcc agc gag atc tcc 1968Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser645650 655tcc atc ctg gag aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc 2016Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr660665 670atc gat gtc tac atg atc atg gtc aag tgc tgg atg ata gac gca gat 2064Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp675 680685agt cgc cca aag ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc 2112Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala690695 700cga gac ccc cag cgc tac ctt gtc att cag ggg gat gaa aga atg cat 2160Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His705710715 720ttg cca agt cct aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa 2208Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu
725 730735gaa gac atg gac gac gtg gtg gat gcc gac gag tac ctc atc cca cag 2256Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln740745750cag ggc ttc ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc 2304Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser755760 765tct ctg agt gca acc agc aac aat tcc acc gtg gct tgc att gat aga 2352Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg770775780aat ggg ctg caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga 2400Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg785790 795 800tac agc tca gac ccc aca ggc gcc ttg act gag gac agc ata gac gac 2448Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp805810 815acc ttc ctc cca gtg cct gaa tac ata aac cag tcc gtt ccc aaa agg 2496Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg820 825 830ccc gct ggc tct gtg cag aat cct gtc tat cac aat cag cct ctg aac 2544Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn835 840845ccc gcg ccc agc aga gac cca cac tac cag gac ccc cac agc act gca 2592Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala850855 860gtg ggc aac ccc gag tat ctc aac act gtc cag ccc acc tgt gtc aac 2640Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn865 870 875 880agc aca ttc gac agc cct gcc cac tgg gcc cag aaa ggc agc cac caa 2688Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln885890 895att agc ctg gac aac cct gac tac cag cag gac ttc ttt ccc aag gaa 2736Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu900905910gcc aag cca aat ggc atc ttt aag ggc tcc aca gct gaa aat gca gaa 2784Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu915 920925tac cta agg gtc gcg cca caa agc agt gaa ttt att gga gca tga 2829Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala930935 94021012211942212PRT213人(homo sapiens)
40012Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val20 25 30Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr35 4045Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro5055 60Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser65 70 75 80Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser85 90 95Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser100 105 110Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys115 120125Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu130 135140Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly145150155160Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn165 170175Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp180185190Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn195 200205Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser210215 220Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala225230235 240Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val
245 250 255Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn260 265 270Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile275 280285Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr290295300Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly305 310315 320Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn325 330 335Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys340 345 350His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys355360365Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly370 375380Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met385390395 400Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln405 410415Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn420 425430Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys435440 445Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile450 455460Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg465 470 475 480Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr485 490 495Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile
500 505 510Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly515520 525Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln530 535 540Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu545550555 560Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu565570 575Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys580 585 590Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val595 600 605Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr610615620His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met625 630 635640Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser645650 655Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr660665 670Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp675 680685Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala690695 700Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His705710 715 720Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu725730735Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln740 745 750Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser
755 760765Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg770775780Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg785790 795800Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp805810 815Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg820 825 830Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn835840 845Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala850 855860Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn865 870 875 880Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln885890 895Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu900905910Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu915 920925TVr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala930935 940210132113633212DNA213小鼠(Mus musculus)220
221CDS222(1)..(3630)223
40013atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc 48Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa 96Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt 144Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac 192Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag 240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg 288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt 336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac 384Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg 432Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat 480Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg 528Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca 576Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag 624Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195200 205aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210215 220ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240
aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305 310315 320gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495
aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550 555560gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615620acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tgg cca 1920Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625 630635640tct ggg cca aag ata cca tct att gcc act ggg att gtg ggt ggc ctc 1968Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly Leu645 650 655ctc ttc ata gtg gtg gtg gcc ctt ggg att ggc cta ttc atg cga aga 2016Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg660 665 670cgt cac att gtt cga aag cgt aca cta cgc cgc ctg ctt caa gag aga 2064Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg675 680685gag ctc gtg gaa cct ctc aca ccc agc gga gaa gct cca aac caa gcc 2112Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala690695 700cac ttg agg ata tta aag gaa aca gaa ttc aaa aag atc aaa gtt ctg 2160His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu705 710715 720ggt tcg gga gca ttt ggc aca gtg tat aag ggt ctc tgg atc cca gaa 2208Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu725 730 735ggt gag aaa gta aaa atc ccg gtg gcc atc aag gag tta aga gaa gcc 2256Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala740 745 750
aca tct cca aaa gcc aac aaa gaa atc ctt gac gaa gcc tat gtg atg 2304Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met755 760 765gct agt gtg gac aac cct cat gta tgc cgc ctc ctg ggc atc tgt ctg 2352Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu770775 780acc tcc act gtc cag ctc att aca cag ctc atg ccc tac ggt tgc ctc 2400Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu785 790 795800ctg gac tac gtc cga gaa cac aag gac aac att ggc tcc cag tac ctc 2448Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu805 810 815ctc aac tgg tgt gtg cag att gca aag ggc atg aac tac ctg gaa gat 2496Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp820 825 830cgg cgt ttg gtg cac cgt gac ttg gca gcc agg aat gta ctg gtg aag 2544Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys835 840845aca cca cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctt 2592Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu850 855 860ggt gct gaa gag aaa gaa tat cat gcc gag ggg ggc aaa gtg cct atc 2640Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile865 870 875 880aag tgg atg gct ttg gaa tca att tta cac cga att tat aca cac caa 2688Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln885890 895agt gat gtc tgg agc tat ggt gtc act gtg tgg gaa ctg atg acc ttt 2736Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe900 905 910ggg tcc aag cct tat gat gga atc cca gca agt gac atc tca tcc atc 2784Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile Ser Ser Ile915 920 925cta gag aaa gga gaa cgc ctt cca cag cca cct atc tgc acc atc gat 2832Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp930935940gtc tac atg atc atg gtc aag tgc tgg atg ata gat gct gat agc cgc 2880Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg945 950 955960cca aag ttc cga gag ttg att ctt gaa ttc tcc aaa atg gcc cga gac 2928Pro Lys Phe Arg Glu Leu Ile Leu Glu Phe Ser Lys Met Ala Arg Asp965970975cca cag cgc tac ctt gtt atc cag ggg gat gaa aga atg cat ttg cca 2976Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro980 985 990agc cct aca gac tcc aac ttt tac cga gcc ctg atg gat gaa gag gac 3024Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp9951000 1005
atg gag gat gta gtt gat gct gat gag tat ctt acc cca cag caa3069Met Glu Asp Val Val Asp Ala Asp Glu Tyr Leu Thr Pro Gln Gln101010151020ggc ttc ttc aac agc ccg tcc acg tcg agg act ccc ctc ttg agt3114Gly Phe Phe Asn Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser1025 10301035tct ctg agt gca act agc aac aat tcc act gtg gct tgc att aat3159Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn1040 10451050aga aat ggg agc tgc cgt gtc aaa gaa gac gcc ttc ttg cag cgg3204Arg Asn Gly Ser Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg1055 10601065tac agc tcc gac ccc aca ggt gct gta aca gag gac aac ata gat3249Tyr Ser Ser Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp1070 10751080gac gca ttc ctc cct gta cct gaa tat gta aac caa tct gtt ccc3294Asp Ala Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro1085 10901095aag agg cca gca ggc tct gtg cag aac cct gtc tat cac aat cag3339Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln1100 11051110ccc ctg cat cca gct cct gga aga gac ctg cat tat caa aat ccc3384Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro1115 11201125cac agc aat gca gtg ggc aac cct gag tat ctc aac act gcc cag3429His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln1130 11351140cct acc tgt ctc agt agt ggg ttt aac agc cct gca ctc tgg atc3474Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu Trp Ile1145 11501155cag aaa ggc agt cac caa atg agc cta gac aac cct gac tac cag3519Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro Asp Tyr Gln1160 11651170cag gac ttc ttc ccc aag gaa acc aag cca aat ggc ata ttt aag3564Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn Gly Ile Phe Lys1175 11801185ggc ccc aca gct gaa aat gca gag tac cta cgg gtg gca cct cca3609Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Pro1190 11951200agc agc gag ttt att gga gca tga 3633Ser Ser Glu Phe Ile Gly Ala1205 1210210142111210212PRT213小鼠(Mus musculus)
40014Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 1015Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn50 5560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195 200205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245 250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305310 315320Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495
Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595600 605Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615 620Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625 630 635 640Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly Leu645 650 655Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg660 665 670Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg675 680685Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala690695 700His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu705 710715 720Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu725730 735Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala740 745 750
Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met755 760 765Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu770775 780Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu785 790 795800Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu805 810 815Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp820 825 830Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys835 840845Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu850 855 860Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile865 870 875 880Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln885890 895Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe900 905 910Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile Ser Ser Ile915 920 925Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp930935940Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg945 950955 960Pro Lys Phe Arg Glu Leu Ile Leu Glu Phe Ser Lys Met Ala Arg Asp965 970 975Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro980 985 990Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp9951000 1005
Met Glu Asp Val Val Asp Ala Asp Glu Tyr Leu Thr Pro Gln Gln101010151020Gly Phe Phe Asn Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser102510301035Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn104010451050Arg Asn Gly Ser Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg105510601065Tyr Ser Ser Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp107010751080Asp Ala Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro108510901095Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln110011051110Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro111511201125His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln113011351140Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu Trp Ile114511501155Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro Asp Tyr Gln116011651170Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn Gly Ile Phe Lys117511801185Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Pro119011951200Ser Ser Glu Phe Ile Gly Ala1205 1210210152111932212DNA213小鼠(Mus musculus)
220
221CDS222(1)..(1929)223
-40015atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc48Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa96Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt144Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe354045ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac192Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 7580acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac384Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg432Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat480Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg528Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca576Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag624Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln
195 200205aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225230235240aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260265 270acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275 280285gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290295 300ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305310 315320gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375380cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385 390395400ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420425 430cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455460gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555560gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776Asp Asn Cys Ile Gln Cys Ala HisTyr Ile Asp Gly Pro His Cys Val580 585590aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595600 605aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615 620acc tat gga gta atg gtt cct gaa atg ttg ctc caa agt atc att tta 1920Thr Tyr Gly Val Met Val Pro Glu Met Leu Leu Gln Ser Ile Ile Leu625 630635640aaa cct att tga 1932Lys Pro Ile21016211643212PRT213小鼠(Mus musculus)40016Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr
1 5 10 15Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 7580Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185190Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195 200205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305 310315320Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405 410415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465 470475 480Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485 490 495Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn
515 520 525Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615 620Thr Tyr Gly Val Met Val Pro Glu Met Leu Leu Gln Ser Ile Ile Leu625 630635640Lys Pro Ile210172111968212DNA213小鼠(Mus musculus)220
221CDS222(1)..(1965)223
40017atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc48Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa96Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30
ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt144Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe35 4045ctg agc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac192Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560ttg gaa att acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag240Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 7075 80acc atc cag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg288Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95gag aga atc cct ttg gag aac ctg cag atc atc agg gga aat gct ctt336Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110tat gaa aac acc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac384Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125aga act ggg ctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg432Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130135140att ggt gct gtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat480Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150155160act atc cag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg528Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175tca atg gac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca576Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190agc tgt ccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag624Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195200 205aaa ttg acc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt672Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220ggc agg tcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt720Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240aca ggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat768Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255gag gcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc816Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270acc acc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt864Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285
gcc acc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300ggc tca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305310 315320gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Set Ile Asn340 345350gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405 410 415ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530 535540
gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610615620acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tgg cca 1920Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625630 635640tct ggg tac gtt caa tgg cag tgg atc tta aag acc ttt tgg atc taa 1968Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile645 650 65521018211655212PRT213小鼠(Mus musculus)40018Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr1 5 10 15Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe354045Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn505560Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu100 105 110Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn115120 125Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135140Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp145 150 155 160Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170175Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro180185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195 200205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg210 215 220Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp245250255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro260 265270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly275280285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His290 295300Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu305 310315320Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val325330335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn340 345350
Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp355 360365Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr370 375 380Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu385390395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450455 460Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465470475 480Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu485490 495Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser500 505510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn515520525Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly530535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545550555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605
Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615620Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625 630635640Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile645 650 655210192111962212DNA213雞220
221CDS222(1)..(1962)223
40019atg ggt gta cgc agc ccc ctg tcc gcc tct ggg cct cgc ggg gcc gct 48Met Gly Val Arg Ser Pro Leu Ser Ala Ser Gly Pro Arg Gly Ala Ala1 5 10 15gtc ctg gtg cta ctg ctg ctg ggc gtc gcg ctg tgt tcc gcc gtg gag 96Val Leu Val Leu Leu Leu Leu Gly Val Ala Leu Cys Ser Ala Val Glu20 25 30gag aag aaa gtt tgt caa ggg aca aat aac aag ttg acc cag ctg ggg 144Glu Lys Lys Val Cys Gln Gly Thr Asn Asn Lys Leu Thr Gln Leu Gly3540 45cac gtg gaa gac cat ttc acc agc ctg cag aga atg tac aac aac tgc 192His Val Glu Asp His Phe Thr Ser Leu Gln Arg Met Tyr Asn Asn Cys50 55 60gaa gtg gta ctg a9t aac ctg gag att acc tac gtg gag cat aat cgc 240Glu Val Val Leu Ser Asn Leu Glu Ile Thr Tyr Val Glu His Asn Arg65 70 75 80gat ctc acc ttc ctt aag acc ata cag gag gtt gca ggc tat gtg ctc 288Asp Leu Thr Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu85 90 95att gcg ctt aac atg gtg gac gtc att ccc tta gaa aac ctc cag att 336Ile Ala Leu Asn Met Val Asp Val Ile Pro Leu Glu Asn Leu Gln Ile100105 110atc cga ggg aat gtg ctt tat gac aac tct ttt gcc ctg gca gtt tta 384Ile Arg Gly Asn Val Leu Tyr Asp Asn Ser Phe Ala Leu Ala Val Leu
115 120125tcc aat tac cac atg aat aaa acc cag gga ctt cga gag ctg cca atg432Ser Asn Tyr His Met Asn Lys Thr Gln Gly Leu Arg Glu Leu Pro Met130135140aaa cgg cta tca gaa att ctc aat gga ggt gtt aaa atc agc aac aac480Lys Arg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile Ser Asn Asn145150 155 160ccc aaa ctg tgc aac atg gac act gtt ctc tgg aat gac atc att gat528Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn Asp Ile Ile Asp165 170 175aca agc agg aag cct ctc aca gta ctt gac ttt gca agc aat ctt tct576Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala Ser Asn Leu Ser180185190tct tgt cca aaa tgc cat ccg aac tgc aca gaa gac cac tgc tgg ggt624Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp His Cys Trp Gly195200205gct ggt gaa cag aac tgc cag act tta aca aaa gtc atc tgt gcc cag672Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val Ile Cys Ala Gln210215220caa tgc tct ggc cgg tgc aga gga aag gtg ccc agt gac tgc tgc cac720Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser Asp Cys Cys His225230 235240aat cag tgt gct gca ggg tgc aca gga cct cgg gag agt gac tgc ctg768Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu245 250 255gca tgc cgc aag ttt cgg gat gat gct acc tgc aag gac aca tgt ccc816Ala Cys Arg Lys Phe Arg Asp Asp Ala Thr Cys Lys Asp Thr Cys Pro260265270cca ctg gtc ctc tat aac ccc acc acc tat caa atg gat gtc aac cct864Pro Leu Val Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro275 280285gag gga aaa tac agc ttt gga gcc act tgt gtg agg gaa tgt cca cac912Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Arg Glu Cys Pro His290 295300aac tat gtg gtg aca gat cat ggc tcc tgc gtt cgc tcg tgt aat act960Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ser Cys Asn Thr305 310315320gat act tac gaa gtg gaa gaa aat ggt gtt cgg aag tgt aaa aaa tgt 1008Asp Thr Tyr Glu Val Glu Glu Asn Gly Val Arg Lys Cys Lys Lys Cys325 330335gat ggg cta tgt agc aaa gtg tgc aat ggc att gga ata ggt gaa ctt 1056Asp Gly Leu Cys Ser Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Leu340345350aaa ggg atc cta tcc ata aat gcc aca aac atc gac tcc ttc aaa aac 1104Lys Gly Ile Leu Ser Ile Asn Ala Thr Asn Ile Asp Ser Phe Lys Asn355 360365tgt acg aag atc aat ggg gat gtc agc att ctt cct gtt gca ttt cta 1152Cys Thr Lys Ile Asn Gly Asp Val Ser Ile Leu Pro Val Ala Phe Leu
370 375 380ggg gat gcc ttc aca aag aca cta ccc ctt gac cct aag aag ctg gat 1200Gly Asp Ala Phe Thr Lys Thr Leu Pro Leu Asp Pro Lys Lys Leu Asp385390 395400gtc ttt aga aca gtc aaa gaa ata tca gga ttt ttg ttg att cag gcc 1248Val Phe Arg Thr Val Lys Glu Ile Ser Gly Phe Leu Leu Ile Gln Ala405 410 415tgg cct gat aat gct act gat ctc tat gct ttt gaa aat ctg gag att 1296Trp Pro Asp Asn Ala Thr Asp Leu Tyr Ala Phe Glu Asn Leu Glu Ile420 425430atc cga ggc cga acc aag cag cac ggc cag tat tcc ctt gct gtt gtt 1344Ile Arg Gly Arg Thr Lys Gln His Gly Gln Tyr Ser Leu Ala Val Val435 440445aac ttg aaa ata cag tcg ttg ggg ctg cgc tcc ctc aag gaa ata agt 1392Asn Leu Lys Ile Gln Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser450 455460gat gga gac att gcc att atg aag aac aag aac ctc tgc tat gct gac 1440Asp Gly Asp Ile Ala Ile Met Lys Asn Lys Asn Leu Cys Tyr Ala Asp465 470 475480acc atg aac tgg cgc agc ttg ttt gct act cag agt cag aaa aca aaa 1488Thr Met Asn Trp Arg Ser Leu Phe Ala Thr Gln Ser Gln Lys Thr Lys485490495att ata cag aac aga aat aaa aat gat tgt act gct gac agg cat gtg 1536Ile Ile Gln Asn Arg Asn Lys Asn Asp Cys Thr Ala Asp Arg His Val500505 510tgt gac ccg ctg tgc tcg gac gtg ggc tgc tgg ggc cca ggg ccc ttc 1584Cys Asp Pro Leu Cys Ser Asp Val Gly Cys Trp Gly Pro Gly Pro Phe515520525cac tgc ttt tcc tgc agg ttt ttc agt cgc cag aag gag tgt gta aaa 1632His Cys Phe Ser Cys Arg Phe Phe Ser Arg Gln Lys Glu Cys Val Lys530 535540cag tgc aac atc ctg caa ggg gag cca cgt gag ttt gaa aga gac tcc 1680Gln Cys Asn Ile Leu Gln Gly Glu Pro Arg Glu Phe Glu Arg Asp Ser545 550555560aaa tgc ctc ccc tgc cac tca gag tgt ctg gta cag aac tcc act gca 1728Lys Cys Leu Pro Cys His Ser Glu Cys Leu Val Gln Asn Ser Thr Ala565 570575tac aac aca acc tgc tct gga ccg ggc cca gac cac tgc atg aag tgt 1776Tyr Asn Thr Thr Cys Ser Gly Pro Gly Pro Asp His Cys Met Lys Cys580585 590gcc cat ttt ata gat ggt ccc cac tgt gtg aag gcc tgc ccc gct ggg 1824Ala His Phe Ile Asp Gly Pro His Cys Val Lys Ala Cys Pro Ala Gly595 600605gtc ctg ggt gag aat gat acc ctg gtc tgg aag tat gca gat gcc aat 1872Val Leu Gly Glu Asn Asp Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn610615620gct gtt tgc cag ctc tgc cat cca aac tgt aca cga ggg tgc aaa ggg 1920Ala Val Cys Gln Leu Cys His Pro Asn Cys Thr Arg Gly Cys Lys Gly
625 630 635 640cca ggt ctt gaa gga tgt cca aat ggc tcc aaa act cca tct1962Pro Gly Leu Glu Gly Cys Pro Asn Gly Ser Lys Thr Pro Ser645 65021020211654212PRT213雞40020Met Gly Val Arg Ser Pro Leu Ser Ala Ser Gly Pro Arg Gly Ala Ala1 5 1015Val Leu Val Leu Leu Leu Leu Gly Val Ala Leu Cys Ser Ala Val Glu20 25 30Glu Lys Lys Val Cys Gln Gly Thr Asn Asn Lys Leu Thr Gln Leu Gly3540 45His Val Glu Asp His Phe Thr Ser Leu Gln Arg Met Tyr Asn Asn Cys50 5560Glu Val Val Leu Ser Asn Leu Glu Ile Thr Tyr Val Glu His Asn Arg65 7075 80Asp Leu Thr Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu85 90 95Ile Ala Leu Asn Met Val Asp Val Ile Pro Leu Glu Asn Leu Gln Ile100105 110Ile Arg Gly Asn Val Leu Tyr Asp Asn Ser Phe Ala Leu Ala Val Leu115120125Ser Asn Tyr His Met Asn Lys Thr Gln Gly Leu Arg Glu Leu Pro Met130135140Lys Arg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile Ser Asn Asn145150 155 160Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn Asp Ile Ile Asp165 170 175Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala Ser Asn Leu Ser
180 185 190Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp His Cys Trp Gly195200 205Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val Ile Cys Ala Gln210215220Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser Asp Cys Cys His225230235 240Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu245 250 255Ala Cys Arg Lys Phe Arg Asp Asp Ala Thr Cys Lys Asp Thr Cys Pro260265270Pro Leu Val Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro275 280285Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Arg Glu Cys Pro His290 295300Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ser Cys Asn Thr305 310315320Asp Thr Tyr Glu Val Glu Glu Asn Gly Val Arg Lys Cys Lys Lys Cys325330 335Asp Gly Leu Cys Ser Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Leu340345350Lys Gly Ile Leu Ser Ile Asn Ala Thr Asn Ile Asp Ser Phe Lys Asn355 360365Cys Thr Lys Ile Asn Gly Asp Val Ser Ile Leu Pro Val Ala Phe Leu370 375380Gly Asp Ala Phe Thr Lys Thr Leu Pro Leu Asp Pro Lys Lys Leu Asp385390 395400Val Phe Arg Thr Val Lys Glu Ile Ser Gly Phe Leu Leu Ile Gln Ala405 410415Trp Pro Asp Asn Ala Thr Asp Leu Tyr Ala Phe Glu Asn Leu Glu Ile420 425430Ile Arg Gly Arg Thr Lys Gln His Gly Gln Tyr Ser Leu Ala Val Val
435 440445Asn Leu Lys Ile Gln Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser450455 460Asp Gly Asp Ile Ala Ile Met Lys Asn Lys Asn Leu Cys Tyr Ala Asp465 470 475 480Thr Met Asn Trp Arg Ser Leu Phe Ala Thr Gln Ser Gln Lys Thr Lys485 490 495Ile Ile Gln Asn Arg Asn Lys Asn Asp Cys Thr Ala Asp Arg His Val500 505510Cys Asp Pro Leu Cys Ser Asp Val Gly Cys Trp Gly Pro Gly Pro Phe515520525His Cys Phe Ser Cys Arg Phe Phe Ser Arg Gln Lys Glu Cys Val Lys530535 540Gln Cys Asn Ile Leu Gln Gly Glu Pro Arg Glu Phe Glu Arg Asp Ser545 550555560Lys Cys Leu Pro Cys His Ser Glu Cys Leu Val Gln Asn Ser Thr Ala565570575Tyr Asn Thr Thr Cys Ser Gly Pro Gly Pro Asp His Cys Met Lys Cys580 585590Ala His Phe Ile Asp Gly Pro His Cys Val Lys Ala Cys Pro Ala Gly595 600605Val Leu Gly Glu Asn Asp Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn610615620Ala Val Cys Gln Leu Cys His Pro Asn Cys Thr Arg Gly Cys Lys Gly625 630635640Pro Gly Leu Glu Gly Cys Pro Asn Gly Ser Lys Thr Pro Ser645 650210212114134212DNA213果蠅(Drosophila melanogaster)
220
221CDS222(1)..(3630)223
40021atg atg att atc agc atg tgg atg agc ata tcg cga gga ttg tgg gac48Met Met Ile Ile Ser Met Trp Met Ser Ile Ser Arg Gly Leu Trp Asp1 5 10 15agc agc tcc atc ttg tcg gtg ctg ctg atc ctc gcc tgc atg gca tcc96Ser Ser Ser Ile Leu Ser Val Leu Leu Ile Leu Ala Cys Met Ala Ser20 25 30atc acc aca agc tca tcg gtc agc aat gcc ggc tat gtg gat aat ggc 144Ile Thr Thr Ser Ser Ser Val Ser Asn Ala Gly Tyr Val Asp Asn Gly35 40 45aat atg aaa gtc tgc atc ggc act aaa tct cgg ctc tcc gtg ccc tcc 192Asn Met Lys Val Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro Ser5055 60aac aag gaa cat cat tac cga aac ctc aga gat cgg tac acg aac tgt 240Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn Cys65 70 7580acg tat gtg gat ggc aac ttg aaa ctg acc tgg cta ccc aac gag aat 288Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu Asn85 9095ttg gac ctc agc ttc cta gac aac ata cgg gag gtc acc ggc tat att 336Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr Ile100105110ctg atc agt cat gtg gac gtt aag aaa gtg gtg ttt ccc aaa cta caa 384Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu Gln115 120125atc att cgc gga cgc acg ctg ttc agc tta tcc gtg gag gag gag aag 432Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu Lys130 135 140tat gcc ttg ttc gtc act tat tcc aaa atg tac acg ctg gag att ccc 480Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile Pro145 150 155 160gat cta cgc gat gtc tta aat ggc caa gtg ggc ttc cac aac aac tac 528Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn Tyr165170 175aat ctc tgc cac atg cga acg atc cag tgg tcg gag att gta tcc aac 576Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser Asn180185 190ggc acg gat gca tac tac aac tac gac ttt act gct ccg gag cgc gag 624Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg Glu195 200205
tgt ccc aag tgc cac gag agc tgc acg cac gga tgt tgg ggc gag ggt 672Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu Gly210215220ccc aag aat tgc cag aag ttc agc aag ctc acc tgc tcg cca cag tgt 720Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln Cys225230235240gcc gga ggt cgt tgc tat gga cca aag ccg cgg gag tgt tgt cac ctc 768Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His Leu245250255ttc tgc gcc gga gga tgc act ggt ccc acg caa aag gat tgc atc gcc 816Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile Ala260 265270tgc aag aac ttc ttc gac gag gca gta tca aag gag gaa tgc ccg ccc 864Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro Pro275280 285atg cgc aag tac aat ccc acc acc tat gtt ctt gaa acg aat cct gag 912Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro Glu290295300gga aag tat gcc tat ggt gcc acc tgc gtc aag gag tgt ccc ggt cat 960Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly His305 310 315320ctg ttg cgg gat aat ggc gcc tgc gtg cgc agc tgt ccc cag gac aag 1008Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp Lys325 330335atg gac aag ggg ggc gag tgt gtg ccc tgc aat gga ccg tgc ccc aaa 1056Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro Lys340345350acc tgc ccg ggc gtt act gtc ctg cat gcc ggc aac att gac tcg ttc 1104Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser Phe355 360 365cgg aat tgt acg gtg atc gat ggc aac att cgc att ttg gat cag acc 1152Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln Thr370 375 380ttc tcg ggc ttc cag gat gtc tat gcc aac tac acg atg gga cca cga 1200Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro Arg385390395 400tac ata ccg ctg gat ccc gag cga cgg gag gtg ttc tcc acg gtg aag 1248Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val Lys405410415gag atc acc ggg tat ctg aat atc gag gga acc cac ccg cag ttc cgg 1296Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe Arg420425 430aat ctg tcg tac ttt cgc aat ctg gaa aca att cat ggc cgc cag ctg 1344Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln Leu435440445atg gag agc atg ttt gcc gct ttg gcg atc gtt aag tca tcc ctg tac 1392Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr450 455 460
agc ctg gag atg cgc aat ctg aag cag att agt tcc ggc agt gtg gtc 1440Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val Val465470475 480atc cag cat aat aga gac ctc tgc tac gta agc aat atc cgt tgg ccg 1488Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp Pro485490495gcc att cag aag gag ccc gaa cag aag gtg tgg gtc aac gag aat ctc 1536Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn Leu500 505510agg gcg gat cta tgc gag aaa aat gga acc att tgc tcg gat cag tgc 1584Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln Cys515 520 525aac gag gac ggc tgc tgg gga gct ggc acg gat cag tgc ctt acc tgc 1632Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr Cys530 535 540aag aac ttc aat ttc aat ggc acc tgc atc gcc gac tgt ggt tat ata 1680Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr Ile545550555 560tcc aat gcc tac aag ttt gac aat aga acg tgc aag ata tgc cat cca 1728Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His Pro565570575gag tgc cgg act tgc aat gga gct gga gca gat cac tgc cag gag tgc 1776Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu Cys580585 590gtc cat gtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag aac 1824Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys Asn595600 605aag tac aac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc gat 1872Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys Asp610615620gga tgc act ggg ccc aag gac acc atc ggc att gga gcg tgt acg acg 1920Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr625 630 635 640tgc aat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc ctg 1968Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu645 650655ctg aag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg cat 2016Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His660 665 670ccg caa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt tgc 2064Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys675 680685cga aag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac cat 2112Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His690695700gaa cag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag tgc 2160Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys705 710715720
gag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc gag 2208Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg Glu725730 735tgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt gcc 2256Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly Ala740 745 750gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat gag 2304Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn Glu755760765acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag tgt 2352Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys Cys770 775 780ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga ccc 2400Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly Pro785790 795800tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat ctg 2448Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn Leu805 810815gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg acg 2496Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro Thr820 825 830atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa aag 2544Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln Lys835 840 845gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc gag 2592Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys Glu850855 860gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc aag 2640Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys865870875 880ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt gga 2688Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly885 890895atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag ggt 2736Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly900 905 910gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc aca 2784Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu LVs Ser Thr915 920 925ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg gcc 2832Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala930 935 940tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg tcc 2880Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser945 950 955 960tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc ctg ttg 2928Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys Leu Leu965 970975
gac tat gtg cga aat aac cgg gac aag atc ggc tct aag gct ctg ctc 2976Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys Ala Leu Leu980985 990aac tgg agc acg caa atc gcc aag ggc atg tcg tat ctg gag gag aag 3024Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Lys995 10001005cga ctg gtc cac aga gac ttg gct gcc cgc aat gtc ctg gtg cag 3069Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gln101010151020act ccc tcg ctg gtg aag atcacc gac ttt ggg ctg gcc aag ttg 3114Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu1025 10301035ctg agc agc gat tcc aat gag tac aag gct gct ggc ggc aag atg 3159Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala Gly Gly Lys Met1040 10451050ccc atc aag tgg ttg gca ctg gag tgc atc cgc aat cgt gta ttc 3204Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg Asn Arg Val Phe1055 10601065acc agc aag tcc gat gtc tgg gcc ttt ggt gtg acc att tgg gaa 3249Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val Thr Ile Trp Glu1070 10751080ctg ctg acc ttt ggc cag cgt cca cac gag aac atc cca gct aag 3294Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn Ile Pro Ala Lys1085 10901095gat att ccc gat ctt att gaa gtc ggt ctg aag ctg gag cag ccg 3339Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys Leu Glu Gln Pro1100 11051110gag att tgt tcg ctg gac att tac tgt aca ctg ctc tcg tgc tgg 3384Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp1115 11201125cac ttg gat gcc gcc atg cgt cca acc ttc aag cag ctg act acg 3429His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr1130 11351140gtc ttt gct gag ttc gcc aga gat ccg ggt cgc tat ctg gcc att 3474Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile1145 11501155ccc ggg gat aag ttc acc cgg ctg ccg gct tac acg agt cag gat 3519Pro Gly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp1160 11651170gag aag gat ctc atc cga aaa ttg gct ccc acc acc gat ggg tcc 3564Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser117511801185gaa gcc att gcg aaa ccc gat gac tac ctg caa ccc aag gca gca 3609Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala1190 11951200cct ggt cct agt cac aga acc gactgcacgg atgagatgcc caagctgaac 3660Pro Gly Pro Ser His Arg Thr12051210
cgctactgca aggatcctag taacaagaat tcgagtaccg gagacgatga gagggattcg 3720agtgcccggg aagtgggcgt gggtaatctg cgcctcgatc taccagtcga tgaggatgat 3780tatctgatgc ccacttgcca accgggtccc aacaacaaca acaacatgaa taatcccaat 3840caaaacaata tggcagctgt gggcgtggct gccggctaca tggatctcat cggagtgccc 3900gttagtgtgg acaatccgga gtatctgcta aacgcgcaga cactgggagt tggggagtcg 3960ccgataccca cccagaccat cgggataccg gtgatgggag gcccgggcac catggaggtc 4020aaggtgccaa tgccaggcag tgagccaacc agctccgatc acgagtacta caatgatacc 4080caacgggagt tgcagccact gcatcgaaac cgcaacacgg agacgagggt gtag4134210222111210212PRT213果蠅(Drosophila melanogaster)40022Met Met Ile Ile Ser Met Trp Met Ser Ile Ser Arg Gly Leu Trp Asp1 5 10 15Ser Ser Ser Ile Leu Ser Val Leu Leu Ile Leu Ala Cys Met Ala Ser20 25 30Ile Thr Thr Ser Ser Ser Val Ser Asn Ala Gly Tyr Val Asp Asn Gly35 40 45Asn Met Lys Val Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro Ser5055 60Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn Cys65 70 7580Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu Asn85 9095Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr Ile100 105 110Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu Gln115120 125Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu Lys130 135 140
Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile Pro145 150155160Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn Tyr165170 175Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser Asn180185 190Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg Glu195 200205Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu Gly210 215220Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln Cys225230235240Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His Leu245 250255Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile Ala260 265270Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro Pro275280 285Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro Glu290295300Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly His305 310 315320Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp Lys325 330335Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro Lys340345350Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser Phe355 360 365Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln Thr370 375 380Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro Arg385390395 400
Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val Lys405410415Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe Arg420 425 430Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln Leu435440445Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr450 455 460Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val Val465470475480Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp Pro485 490 495Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn Leu500 505 510Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln Cys515520 525Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr Cys530 535 540Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr Ile545550555 560Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His Pro565570575Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu Cys580585 590Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys Asn595 600605Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys Asp610 615 620Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr625630 635 640Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu645 650 655
Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His660665670Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys675 680685Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His690695700Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys705 710715 720Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg Glu725730 735Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly Ala740 745750Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn Glu755760765Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys Cys770775780Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly Pro785790 795800Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn Leu805 810815Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro Thr820 825 830Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln Lys835840 845Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys Glu850 855 860Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys865870 875 880Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly885 890895Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly900905 910
Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser Thr915 920 925Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala930935940Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser945 950955960Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys Leu Leu965970975Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys Ala Leu Leu980985 990Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Lys995 10001005Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gln101010151020Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu102510301035Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala Gly Gly Lys Met104010451050Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg Asn Arg Val Phe105510601065Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val Thr Ile Trp Glu107010751080Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn Ile Pro Ala Lys108510901095Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys Leu Glu Gln Pro110011051110Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp111511201125His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr113011351140Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile114511501155
Pro Gly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp116011651170Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser117511801185Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala119011951200Pro Gly Pro Ser His Arg Thr12051210210232114281212DNA213果蠅(Drosophila melanogaster)40023atgctgctgc gacggcgcaa cggcccctgc cccttccccc tgctgcttct gctcctggcc 60cactgcattt gcatttggcc cgcgtcggcg gcccgcgatc gctacgcccg ccagaacaat 120cgccagcgcc atcaggatat agatcgcgat cgggatcgag atcgattcct ataccgcagc 180agttcggccc aaaatcgaca gaggggcggg gccaacttcg ccctgggact gggagccaac 240ggagtcacca ttcccaccag tctggaggat aagaacaaga acgagttcgt caaggggaaa 300atctgcatcg gcactaaatc tcggctctcc gtgccctcca acaaggaaca tcattaccga 360aacctcagag atcggtacac gaactgtacg tatgtggatg gcaacttgaa actgacctgg 420ctacccaacg agaatttgga cctcagcttc ctagacaaca tacgggaggt caccggctat 480attctgatca gtcatgtgga cgttaagaaa gtggtgtttc ccaaactaca aatcattcgc 540ggacgcacgc tgttcagctt atccgtggag gaggagaagt atgccttgtt cgtcacttat 600tccaaaatgt acacgctgga gattcccgat ctacgcgatg tcttaaatgg ccaagtgggc 660ttccacaaca actacaatct ctgccacatg cgaacgatcc agtg9tcgga gattgtatcc 720aacggcacgg atgcatacta caactacgac tttactgctc cggagcgcga gtgtcccaag 780tgccacgaga gctgcacgca cggatgttgg ggcgagggtc ccaagaattg ccagaagttc 840agcaagctca cctgctcgcc acagtgtgcc ggaggtcgtt gctatggacc aaagccgcgg 900gagtgttgtc acctcttctg cgccggagga tgcactggtc ccacgcaaaa ggattgcatc 960gcctgcaaga acttcttcga cgaggcagta tcaaaggagg aatgcccgcc catgcgcaag 1020tacaatccca ccacctatgt tcttgaaacg aatcctgagg gaaagtatgc ctatggtgcc 1080
acctgcgtca aggagtgtcc cggtcatctg ttgcgggata atggcgcctg cgtgcgcagc 1140tgtccccagg acaagatgga caaggggggc gagtgtgtgc cctgcaatgg accgtgcccc 1200aaaacctgcc cgggcgttac tgtcctgcat gccggcaaca ttgactcgtt ccggaattgt 1260acggtgatcg atggcaacat tcgcattttg gatcagacct tctcgggctt ccaggatgtc 1320tatgccaact acacgatggg accacgatac ataccgctgg atcccgagcg acgggaggtg 1380ttctccacgg tgaaggagat caccgggtat ctgaatatcg agggaaccca cccgcagttc 1440cggaatctgt cgtactttcg caatctggaa acaattcatg gccgccagct gatggagagc 1500atgtttgccg ctttggcgat cgttaagtca tccctgtaca gcctggagat gcgcaatctg 1560aagcagatta gttccg9cag tgtggtcatc cagcataata gagacctctg ctacgtaagc 1620aatatccgtt ggccggccat tcagaaggag cccgaacaga aggtgtgggt caacgagaat 1680ctcagggcgg atctatgcga gaaaaatgga accatttgct cggatcagtg caacgaggac 1740ggctgctggg gagctggcac ggatcagtgc cttacctgca agaacttcaa tttcaatggc 1800acctgcatcg ccgactgtgg ttatatatcc aatgcctaca agtttgacaa tagaacgtgc 1860aagatatgcc atccagagtg ccggacttgc aatggagctg gagcagatca ctgccaggag 1920tgcgtccatg tgagggacgg tcagcactgt gtgtccgagt gcccgaagaa caagtacaac 1980gatcgtggtg tctgccgaga gtgccacgcc acctgcgatg gatgcactgg gcccaaggac 2040accatcggca ttggagcgtg tacgacgtgc aatttggcca ttatcaacaa tgacgccaca 2100gtaaaacgct gcctgctgaa ggacgacaag tgccccgatg ggtatttctg ggagtatgtg 2160catccgcaag agcagggatc gctgaagcca ttggccggca gagcagtttg ccgaaagtgc 2220catccccttt gcgagctgtg cacgaactac ggataccatg aacaggtgtg ctccaagtgc 2280acccactaca agcgacggga gcagtgcgag accgagtgtc cggccgatca ctacacggat 2340gaggagcagc gcgagtgctt ccagcgccac ccggaatgca atggttgcac gggtccgggt 2400gccgacgatt gcaagtcttg ccgcaacttt aagttgttcg acgcgaatga gacgggtccc 2460tatgtgaact ccacgatgtt caattgcacc tcgaagtgtc ccttggagat gcgacatgtg 2520aactatcagt acacggccat tggaccctac tgcgcagcta gtccgccgag gagcagcaag 2580ataactgcca atctggatgt gaacatgatc ttcattatca ctggtgctgt tctggtgccg 2640acgatctgca tcctctgcgt ggtcacatac atttgtcggc aaaagcaaaa ggccaagaag 2700gaaacagtca agatgaccat ggctctgtct ggctgcgagg attccgagcc gctgcgtccc 2760tcgaacattg gagccaacct atgcaagttg cgcattgtca aggacgccga gttgcgcaag 2820ggcggagtcc ttggaatggg agcctttgga cgagtgtaca agggcgtttg ggtgccggag 2880ggtgagaacg tcaagattcc agtggccatt aaggagctgc tcaagtccac aggcgccgag 2940tcaagcgaag agttcctccg cgaagcctac atcatggcct ctgaggagca cgttaatctg 3000
ctgaagctcc tggccgtgtg catgtcctca caaatgatgc taatcacgca actgatgccg 3060cttggctgcc tgttggacta tgtgcgaaat aaccgggaca agatcggctc taaggctctg 3120ctcaactgga gcacgcaaat cgccaagggc atgtcgtatc tggaggagaa gcgactggtc 3180cacagagact tggctgcccg caatgtcctg gtgcagactc cctcgctggt gaagatcacc 3240gactttgggc tggccaagtt gctgagcagc gattccaatg agtacaaggc tgctggcggc 3300aagatgccca tcaagtggtt ggcactggag tgcatccgca atcgtgtatt caccagcaag 3360tccgatgtct gggcctttgg tgtgaccatt tgggaactgc tgacctttgg ccagcgtcca 3420cacgagaaca tcccagctaa ggatattccc gatcttattg aagtcggtct gaagctggag 3480cagccggaga tttgttcgct ggacatttac tgtacactgc tctcgtgctg gcacttggat 3540gccgccatgc gtccaacctt caagcagctg actacggtct ttgctgagtt cgccagagat 3600ccgggtcgct atctggccat tcccggggat aagttcaccc ggctgccggc ttacacgagt 3660caggatgaga aggatctcat ccgaaaattg gctcccacca ccgatgggtc cgaagccatt 3720gcgaaacccg atgactacct gcaacccaag gcagcacctg gtcctagtca cagaaccgac 3780tgcacggatg agatgcccaa gctgaaccgc tactgcaagg atcctagtaa caagaattcg 3840agtaccggag acgatgagag ggattcgagt gcccgggaag tgggcgtggg taatctgcgc 3900ctcgatctac cagtcgatga ggatgattat ctgatgccca cttgccaacc gggtcccaac 3960aacaacaaca acatgaataa tcccaatcaa aacaatatgg cagctgtggg cgtggctgcc 4020ggctacatgg atctcatcgg agtgcccgtt agtgtggaca atccggagta tctgctaaac 4080gcgcagacac tgggagttgg ggagtcgccg atacccaccc agaccatcgg gataccggtg 4140atgggaggcc cgggcaccat ggaggtcaag gtgccaatgc caggcagtga gccaaccagc 4200tccgatcacg agtactacaa tgatacccaa cgggagttgc agccactgca tcgaaaccgc 4260aacacggaga cgagggtgta g 4281210242114281212DNA213大鼠(Rattus norvegicus)220
221CDS222(1)..(3627)223
40024atg ctg ctg cga cgg cgc aac ggc ccc tgc ccc ttc ccc ctg ctg ctt 48Met Leu Leu Arg Arg Arg Asn Gly Pro Cys Pro Phe Pro Leu Leu Leu1 5 1015ctg ctc ctg gcc cac tgc att tgc att tgg ccc gcg tcg gcg gcc cgc 96Leu Leu Leu Ala His Cys Ile Cys Ile Trp Pro Ala Ser Ala Ala Arg20 25 30gat cgc tac gcc cgc cag aac aat cgc cag cgc cat cag gat ata gat 144Asp Arg Tyr Ala Arg Gln Asn Asn Arg Gln Arg His Gln Asp Ile Asp35 4045cgc gat cgg gat cga gat cga ttc cta tac cgc agc agt tcg gcc caa 192Arg Asp Arg Asp Arg Asp Arg Phe Leu Tyr Arg Ser Ser Ser Ala Gln50 5560aat cga cag agg ggc ggg gcc aac ttc gcc ctg gga ctg gga gcc aac 240Asn Arg Gln Arg Gly Gly Ala Asn Phe Ala Leu Gly Leu Gly Ala Asn6570 75 80gga gtc acc att ccc acc agt ctg gag gat aag aac aag aac gag ttc 288Gly Val Thr Ile Pro Thr Ser Leu Glu Asp Lys Asn Lys Asn Glu Phe85 9095gtc aag ggg aaa atc tgc atc ggc act aaa tct cgg ctc tcc gtg ccc 336Val Lys Gly Lys Ile Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro100 105 110tcc aac aag gaa cat cat tac cga aac ctc aga gat cgg tac acg aac 384Ser Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn115 120125tgt acg tat gtg gat ggc aac ttg aaa ctg acc tgg cta ccc aac gag 432Cys Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu130135140aat ttg gac ctc agc ttc cta gac aac ata cgg gag gtc acc ggc tat 480Asn Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr145 150155 160att ctg atc agt cat gtg gac gtt aag aaa gtg gtg ttt ccc aaa cta 528Ile Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu165 170 175caa atc att cgc gga cgc acg ctg ttc agc tta tcc gtg gag gag gag 576Gln Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu180 185190aag tat gcc ttg ttc gtc act tat tcc aaa atg tac acg ctg gag att 624Lys Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile195 200 205ccc gat cta cgc gat gtc tta aat ggc caa gtg ggc ttc cac aac aac 672Pro Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn210 215220tac aat ctc tgc cac atg cga acg atc cag tgg tcg gag att gta tcc 720Tyr Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser225230235 240aac ggc acg gat gca tac tac aac tac gac ttt act gct ccg gag cgc 768Asn Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg
245 250255gag tgt ccc aag tgc cac gag agc tgc acg cac gga tgt tgg ggc gag 816Glu Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu260 265270ggt ccc aag aat tgc cag aag ttc agc aag ctc acc tgc tcg cca cag 864Gly Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln275280285tgt gcc gga ggt cgt tgc tat gga cca aag ccg cgg gag tgt tgt cag 912Cys Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His290295 300ctc ttc tgc gcc gga gga tgc act ggt ccc acg caa aag gat tgc atc 960Leu Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile305310315 320gcc tgc aag aac ttc ttc gac gag gca gta tca aag gag gaa tgc ccg 1008Ala Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro325 330 335ccc atg cgc aag tac aat ccc acc acc tat gtt ctt gaa acg aat cct 1056Pro Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro340345 350gag gga aag tat gcc tat ggt gcc acc tgc gtc aag gag tgt ccc ggt 1104Glu Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly355 360 365cat ctg ttg cgg gat aat ggc gcc tgc gtg cgc agc tgt ccc cag gac 1152His Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp370375380aag atg gac aag ggg ggc gag tgt gtg ccc tgc aat gga ccg tgc ccc 1200Lys Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro385390395400aaa acc tgc ccg ggc gtt act gtc ctg cat gcc ggc aac att gac tcg 1248Lys Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser405 410415ttc cgg aat tgt acg gtg atc gat ggc aac att cgc att ttg gat cag 1296Phe Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln420425430acc ttc tcg ggc ttc cag gat gtc tat gcc aac tac acg atg gga cca 1344Thr Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro435440 445cga tac ata ccg ctg gat ccc gag cga cgg gag gtg ttc tcc acg gtg 1392Arg Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val450 455460aag gag atc acc ggg tat ctg aat atc gag gga acc cac ccg cag ttc 1440Lys Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe465 470 475 480cgg aat ctg tcg tac ttt cgc aat ctg gaa aca att cat ggc cgc cag 1488Arg Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln485 490 495ctg atg gag agc atg ttt gcc gct ttg gcg atc gtt aag tca tcc ctg 1536Leu Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu
500 505 510tac agc ctg gag atg cgc aat ctg aag cag att agt tcc ggc agt gtg 1584Tyr Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val515520525gtc atc cag cat aat aga gac ctc tgc tac gta agc aat atc cgt tgg 1632Val Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp530 535540ccg gcc att cag aag gag ccc gaa cag aag gtg tgg gtc aac gag aat 1680Pro Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn545 550555 560ctc agg gcg gat cta tgc gag aaa aat gga acc att tgc tcg gat cag 1728Leu Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln565570575tgc aac gag gac ggc tgc tgg gga gct ggc acg gat cag tgc ctt acc 1776Cys Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr580585 590tgc aag aac ttc aat ttc aat ggc acc tgc atc gcc gac tgt ggt tat 1824Cys Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr595600605ata tcc aat gcc tac aag ttt gac aat aga acg tgc aag ata tgc cat 1872Ile Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His610 615620cca gag tgc cgg act tgc aat gga gct gga gca gat cac tgc cag gag 1920Pro Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu625630 635640tgc gtc cat gtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag 1968Cys Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys645 650655aac aag tac aac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc 2016Asn Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys660665670gat gga tgc act ggg ccc aag gac acc atc ggc att gga gcg tgt acg 2064Asp Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr675 680685acg tgc aat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc 2112Thr Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys690695 700ctg ctg aag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg 2160Leu Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val705710715720cat ccg caa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt 2208His Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val725 730735tgc cga aag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac 2256Cys Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr740 745 750cat gaa cag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag 2304His Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln
755 760 765tgc gag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc 2352Cys Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg770775 780gag tgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt 2400Glu Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly785790 795 800gcc gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat 2448Ala Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn805810 815gag acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag 2496Glu Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys820 825830tgt ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga 2544Cys Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly835840 845ccc tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat 2592Pro Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn850855 860ctg gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg 2640Leu Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro865870 875 880acg atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa 2688Thr Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln885 890 895aag gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc 2736Lys Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys900 905 910gag gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc 2784Glu Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys915 920925aag ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt 2832Lys Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu930 935 940gga atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag 2880Gly Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu945 950955 960ggt gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc 2928Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser965 970 975aca ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg 2976Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met980 985990gcc tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg 3024Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met995 1000 1005tcc tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc 3069Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys
101010151020ctg ttg gac tat gtg cga aat aac cgg gac aag atc ggc tct aag 3114Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys102510301035gct ctg ctc aac tgg agc acg caa atc gcc aag ggc atg tcg tat 3159Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr104010451050ctg gag gag aag cga ctg gtc cac aga gac ttg gct gcc cgc aat 3204Leu Glu Glu Lys Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn105510601065gtc ctg gtg cag act ccc tcg ctg gtg aag atc acc gac ttt ggg 3249Val Leu Val Gln Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly107010751080ctg gcc aag ttg ctg agc agc gat tcc aat gag tac aag gct gct 3294Leu Ala Lys Leu Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala108510901095ggc ggc aag atg ccc atc aag tgg ttg gca ctg gag tgc atc cgc 3339Gly Gly Lys Met Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg110011051110aat cgt gta ttc acc agc aag tcc gat gtc tgg gcc ttt ggt gtg 3384Asn Arg Val Phe Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val111511201125acc att tgg gaa ctg ctg acc ttt ggc cag cgt cca cac gag aac 3429Thr Ile Trp Glu Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn113011351140atc cca gct aag gat att ccc gat ctt att gaa gtc ggt ctg aag 3474Ile Pro Ala Lys Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys114511501155ctg gag cag ccg gag att tgt tcg ctg gac att tac tgt aca ctg 3519Leu Glu Gln Pro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu116011651170ctc tcg tgc tgg cac ttg gat gcc gcc atg cgt cca acc ttc aag 3564Leu Ser Cys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys117511801185cag ctg act acg gtc ttt gct gag ttc gcc aga gat ccg ggt cgc 3609Gln Leu Thr Thr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg119011951200tat ctg gcc att ccc ggg gataagttca cccggctgcc ggcttacacg 3657Tyr Leu Ala Ile Pro Gly1205agtcaggatg agaaggatct catccgaaaa ttggctccca ccaccgatgg gtccgaagcc 3717attgcgaaac ccgatgacta cctgcaaccc aaggcagcac ctggtcctag tcacagaacc 3777gactgcacgg atgagatgcc caagctgaac cgctactgca aggatcctag taacaagaat 3837tcgagtaccg gagacgatga gagggattcg agtgcccggg aagtgggcgt gggtaatctg 3897cgcctcgatc taccagtcga tgaggatgat tatctgatgc ccacttgcca accgggtccc 3957
aacaacaaca acaacatgaa taatcccaat caaaacaata tggcagctgt gggcgtggct 4017gccggctaca tggatctcat cggagtgccc gttagtgtgg acaatccgga gtatctgcta 4077aacgcgcaga cactgggagt tggggagtcg ccgataccca cccagaccat cgggataccg 4137gtgatgggag gcccgggcac catggaggtc aaggtgccaa tgccaggcag tgagccaacc 4197agctccgatc acgagtacta caatgatacc caacgggagt tgcagccact gcatcgaaac 4257cgcaacacgg agacgagggt gtag 4281210252111209212PRT213大鼠(Rattus norvegicus)40025Met Leu Leu Arg Arg Arg Asn Gly Pro Cys Pro Phe Pro Leu Leu Leu1 5 10 15Leu Leu Leu Ala His Cys Ile Cys Ile Trp Pro Ala Ser Ala Ala Arg20 25 30Asp Arg Tyr Ala Arg Gln Asn Asn Arg Gln Arg His Gln Asp Ile Asp35 4045Arg Asp Arg Asp Arg Asp Arg Phe Leu Tyr Arg Ser Ser Ser Ala Gln505560Asn Arg Gln Arg Gly Gly Ala Asn Phe Ala Leu Gly Leu Gly Ala Asn6570 75 80Gly Val Thr Ile Pro Thr Ser Leu Glu Asp Lys Asn Lys Asn Glu Phe85 9095Val Lys Gly Lys Ile Cys Ile Gly Thr Lys Ser Arg Leu Ser Val Pro100 105 110Ser Asn Lys Glu His His Tyr Arg Asn Leu Arg Asp Arg Tyr Thr Asn115 120125Cys Thr Tyr Val Asp Gly Asn Leu Lys Leu Thr Trp Leu Pro Asn Glu130135140Asn Leu Asp Leu Ser Phe Leu Asp Asn Ile Arg Glu Val Thr Gly Tyr145 150155 160
Ile Leu Ile Ser His Val Asp Val Lys Lys Val Val Phe Pro Lys Leu165 170175Gln Ile Ile Arg Gly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu180 185190Lys Tyr Ala Leu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile195 200 205Pro Asp Leu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn210215 220Tyr Asn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser225230235 240Asn Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg245250255Glu Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu260 265270Gly Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln275280 285Cys Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys His290295 300Leu Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp Cys Ile305310315 320Ala Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu Glu Cys Pro325 330 335Pro Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu Glu Thr Asn Pro340345 350Glu Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val Lys Glu Cys Pro Gly355 360 365His Leu Leu Arg Asp Asn Gly Ala Cys Val Arg Ser Cys Pro Gln Asp370375380Lys Met Asp Lys Gly Gly Glu Cys Val Pro Cys Asn Gly Pro Cys Pro385390395400Lys Thr Cys Pro Gly Val Thr Val Leu His Ala Gly Asn Ile Asp Ser405410 415
Phe Arg Asn Cys Thr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln420 425 430Thr Phe Ser Gly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro435440 445Arg Tyr Ile Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val450 455460Lys Glu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe465 470 475 480Arg Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln485 490 495Leu Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu500505510Tyr Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val515520525Val Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp530 535540Pro Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn Glu Asn545 550 555 560Leu Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln565570 575Cys Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr580585 590Cys Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr595600605Ile Ser Asn Ala Tyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His610 615620Pro Glu Cys Arg Thr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu625630 635640Cys Val His Val Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys645650655Asn Lys Tyr Asn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys660665670
Asp Gly Cys Thr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr675 680685Thr Cys Asn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys690695 700Leu Leu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val705710715720His Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val725 730735Cys Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr740745750His Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln755760765Cys Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg770775780Glu Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly785790 795 800Ala Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn805810 815Glu Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys820 825830Cys Pro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly835840 845Pro Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn850 855860Leu Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro865870 875 880Thr Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln885 890 895Lys Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys900 905 910Glu Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys915 920925
Lys Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu930 935 940Gly Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu945 950955 960Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser965 970 975Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met980 985990Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met995 10001005Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys1010 1015 1020Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys1025 1030 1035Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr1040 1045 1050Leu Glu Glu Lys Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn1055 1060 1065Val Leu Val Gln Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly1070 1075 1080Leu Ala Lys Leu Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala1085 1090 1095Gly Gly Lys Met Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg1100 1105 1110Asn Arg Val Phe Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val1115 1120 1125Thr Ile Trp Glu Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn1130 1135 1140Ile Pro Ala Lys Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys1145 1150 1155Leu Glu Gln Pro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu1160 1165 1170
Leu Ser Cys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys117511801185Gln Leu Thr Thr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg119011951200Tyr Leu Ala Ile Pro Gly1205210262113576212DNA213斑馬魚(Danio rerio)220
221CDS222(1)..(3573)223
40026atg gca gga cca act gaa atc gga ttg ttt ttt acc tta ttg ctc tcc 48Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe Thr Leu Leu Leu Ser1 5 1015ggg agc ttc tgc gcg acg ccg gaa aag aaa gtg tgt cag gga gca aac 96Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys Val Cys Gln Gly Ala Asn20 25 30aac aaa ctg act ctt ctg gga acg gtg gaa gac cat tat cag gtt ctg 144Asn Lys Leu Thr Leu Leu Gly Thr Val Glu Asp His Tyr Gln Val Leu35 40 45ctc aga atg tac aga aac tgc act gtg gtt ctg gag aac ctg gaa att 192Leu Arg Met Tyr Arg Asn Cys Thr Val Val Leu Glu Asn Leu Glu Ile505560aca cat ata aca gag aaa tat gac ctg tcc ttc ctc aag agc atc cag 240Thr His Ile Thr Glu Lys Tyr Asp Leu Ser Phe Leu Lys Ser Ile Gln65 70 7580gaa gtt ggt ggc tat gtt ctt atc gcg gtc aat acg gtt tcc aaa atc 288Glu Val Gly Gly Tyr Val Leu Ile Ala Val Asn Thr Val Ser Lys Ile85 90 95cct ctg gag aac ctg cgc atc att cgc gga cac tca ctt tat gaa gac 336Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly His Ser Leu Tyr Glu Asp100 105 110aaa ttt gcc ttg gcc gtc ctg gtg aac ttc aac aac agc atc gaa caa 384Lys Phe Ala Leu Ala Val Leu Val Asn Phe Asn Asn Ser Ile Glu Gln
115 120 125ggc gta aaa gag ttg ccg ctg act agt tta act gaa ata ctt aag ggt 432Gly Val Lys Glu Leu Pro Leu Thr Ser Leu Thr Glu Ile Leu Lys Gly130 135140gga gtc aag ttt tgc agg aac gat tat tta tgt aat gtg ggg acc atc 480Gly Val Lys Phe Cys Arg Asn Asp Tyr Leu Cys Asn Val Gly Thr Ile145 150 155160gag tgg gcc gac atc ctg aac atg aag agc ctg cct aca atc gtg agc 528Glu Trp Ala Asp Ile Leu Asn Met Lys Ser Leu Pro Thr Ile Val Ser165170175cat aat ata agc tat gga aaa aac tgt gga aag tgc gat cca agc tgt 576His Asn Ile Ser Tyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys180 185190ttc aat ggc tcc tgc tgg ggc acc gga ccc gac aag tgc cag aga atg 624Phe Asn Gly Ser Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met195200 205acg aaa gtg atc tgt gcg gag cag tgt tca ggg agg tgt aaa gga ccc 672Thr Lys Val Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro210 215220aga ccc att gac tgc tgt aat gaa cac tgt gct gct gga tgc act gga 720Arg Pro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly225 230 235 240ccc aga cct aca gac tgt ctg gcc tgt aag gac ttc cag gat gaa ggg 768Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp Glu Gly245250255aca tgt aag gac gca tgt ccg cgg ctc atg ctc tac gac cca aac aca 816Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro Asn Thr260 265 270cac cag ctc gcg cca aac cca tat ggg aag tac agc ttt ggg gca acg 864His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly Ala Thr275280 285tgc atc aag aca tgc cca cac aac tat gtg gtg acg gat cac ggg gcc 912Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp His Gly Ala290 295 300tgt gtg aga aca tgc agc cct ggc acc tat gaa gtg gat gag ggt gga 960Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val Asp Glu Gly Gly305 310315320gtt cgc aaa tgt aag agg tgt gaa ggc ctg tgt cca aaa gtg tgc aat 1008Val Arg Lys Cys Lys Arg Cys Glu Gly Leu Cys Pro Lys Val Cys Asn325330335ggg ttg gga atg ggg cct tta gcc aat gtc ctg tca atc aat gcc acc 1056Gly Leu Gly Met Gly Pro Leu Ala Asn Val Leu Ser Ile Asn Ala Thr340 345350aac atc gac tcc ttt gag aac tgc act aaa atc agc ggc aat gtt gcc 1104Asn Ile Asp Ser Phe Glu Asn Cys Thr Lys Ile Ser Gly Asn Val Ala355360365atc ctc agc acc aca ttc aga ggt gac cca cat act aac act tca gga 1152Ile Leu Ser Thr Thr Phe Arg Gly Asp Pro His Thr Asn Thr Ser Gly
370375 380ctg gat cca gca aag ctc agt gta ttg agt act gtc aaa gaa atc act1200Leu Asp Pro Ala Lys Leu Ser Val Leu Ser Thr Val Lys Glu Ile Thr385 390395400ggt tac ctg atg att cag ctg tgg ccg gag agc atg cag tcc ctt agt1248Gly Tyr Leu Met Ile Gln Leu Trp Pro Glu Ser Met Gln Ser Leu Ser405410 415gcc ttc gaa aac ctt gag gtc atc cga gga cgg aca aaa aca caa gga1296Ala Phe Glu Asn Leu Glu Val Ile Arg Gly Arg Thr Lys Thr Gln Gly420425 430acg tac agc ttt gct gtc acc aag acg gcc atc act cat tta ggc atg1344Thr Tyr Ser Phe Ala Val Thr Lys Thr Ala Ile Thr His Leu Gly Met435440 445cgt tct ctg agg gag atc agt gac ggg gac gtg tcc atc gtt aag aat1392Arg Ser Leu Arg Glu Ile Ser Asp Gly Asp Val Ser Ile Val Lys Asn450455 460aag aat ctc tgc tac agc agc cct gaa cac tgg aaa cgc ctc ttc aag1440Lys Asn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys Arg Leu Phe Lys465470 475480tcc aaa caa cag tcg gtc aaa atg att gaa aat atg gat gct gcc acc1488Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn Met Asp Ala Ala Thr485 490495tgc gcc aat cag aac agc aca tgt aat gag atg tgc acg gct gac ggc1536Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met Cys Thr Ala Asp Gly500505510tgc tgg ggt ccc ggc ccc acc atg tgc ttc ggc tgt gag cat tac agc1584Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly Cys Glu His Tyr Ser515 520525cgc gga aaa cac tgc gtg gct tcc tgc aac ctg ctg aat ggt gag ccg1632Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu Leu Asn Gly Glu Pro530535 540cgt gaa tat gag gtc aat aaa aca tgc atg gaa tgc gat cct gaa tgt1680Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu Cys Asp Pro Glu Cys545 550555560ctg ctc atg aat gaa acc cag acc tgc aac ggc cct gga ccc gac aaa1728Leu Leu Met Asn Glu Thr Gln Thr Cys Asn Gly Pro Gly Pro Asp Lys565 570 575tgt aca gtg tgt gca aac tat aaa gac gga ccg cac tgt gtg cat cgc1776Cys Thr Val Cys Ala Asn Tyr Lys Asp Gly Pro His Cys Val His Arg580 585590tgc ccg caa ggt gta cca gga gag aaa gac aca ctc atc tgg aaa tac1824Cys Pro Gln Gly Val Pro Gly Glu Lys Asp Thr Leu Ile Trp Lys Tyr595 600 605gct gac gtg aca cac gtt tgc cag ccc tgt cat gaa aac tgc acc cag1872Ala Asp Val Thr His Val Cys Gln Pro Cys His Glu Asn Cys Thr Gln610 615620gga tgt acg ggg cct gat cta aag gac tgc aaa gat ttc aaa agc tct1920Gly Cys Thr Gly Pro Asp Leu Lys Asp Cys Lys Asp Phe Lys Ser Ser
625 630 635 640ggt ttg ccg atg atc gct gct ggc gtt gtc gga ggt cta ctg gcg ttt 1968Gly Leu Pro Met Ile Ala Ala Gly Val Val Gly Gly Leu Leu Ala Phe645 650 655gtt att ctg gct ctt gga gtg gcc gtt ctc ctg cgc aga cgc cac atc 2016Val Ile Leu Ala Leu Gly Val Ala Val Leu Leu Arg Arg Arg His Ile660665 670cgg agg aag agg act ctg aga cga ctc ctg caa gag aga gag ctt gtg 2064Arg Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val675680685gag cct ctg acc ccc agc ggc gaa gcc ccc aat cag gcc tta ctg cgc 2112Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg690695 700atc ctc aaa gag acg gag ttc aag aag atc aaa gtg ctg ggc tcc ggg 2160Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly705 710 715 720gct ttc ggc act gtg cac aag ggc ctt tgg gtt cca gaa gga gag aat 2208Ala Phe Gly Thr Val His Lys Gly Leu Trp Val Pro Glu Gly Glu Asn725 730 735gtg aag atc cct gtc gcc atc aag gtc tta aga gaa gcc acg tct ccc 2256Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Ala Thr Ser Pro740 745 750aaa gct aac aag gag ata atg gat gag gcg tac gtc atg gcc agt gtt 2304Lys Ala Asn Lys Glu Ile Met Asp Glu Ala Tyr Val Met Ala Ser Val755 760765gag cat ccc cat gtg tgt cgt ctg ctg ggc atc tgc ttg acc tcc aca 2352Glu His Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr770 775780gtg cag ctc atc act cag ctg atg ccc tac ggc tgc ttg ctg gac tat 2400Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp Tyr785 790795 800gtc aga gaa aac aag gac cgc atc ggc tcc cag cac ctg ctc aac tgg 2448Val Arg Glu Asn Lys Asp Arg Ile Gly Ser Gln His Leu Leu Asn Trp805810 815tgc gtg cag atc gct aaa ggt atg aat tat cta gaa gag cgc cat ctt 2496Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Glu Arg His Leu820 825 830gtg cac cga gac ctg gca gca cgt aat gtg ttg gta aag acg cct cag 2544Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln835840845cat gtc aag att acc gat ttc ggc ctc gcc aag ctg tta aac gcg gac 2592His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Asn Ala Asp850 855 860gag aag gag tat cac gca gat gga gga aag gtt cca att aaa tgg atg 2640Glu Lys Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met865 870 875 880gcg ctg gag tcc atc cag cac agg act tac acc cac cag agt gac gtc 2688Ala Leu Glu Ser Ile Gln His Arg Thr Tyr Thr His Gln Ser Asp Val
885 890 895tgg agc tac ggt gtg acc gtc tgg gag ttg atg acg ttt ggg acg aaa 2736Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Thr Lys900 905 910cct tat gat ggg att cct gcc agt gaa atc gcc gga gtt ctg gaa aaa 2784Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ala Gly Val Leu Glu Lys915 920 925gga gaa aga ctt cct caa ccc ccc atc tgc acc att gat gtg tac atg 2832Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met930935 940atc atg gtc aaa tgt tgg atg att gat gct gag agc aga ccc cgc ttc 2880Ile Met Val Lys Cys Trp Met Ile Asp Ala Glu Ser Arg Pro Arg Phe945 950 955 960agg gag ctc atc gca gag ttc act aaa atg gct cgt gac ccg tcc cgc 2928Arg Glu Leu Ile Ala Glu Phe Thr Lys Met Ala Arg Asp Pro Ser Arg965970 975tat ctg gtc att cag ggg gac gac cgc atg cat tta ccc agt cct tct 2976Tyr Leu Val Ile Gln Gly Asp Asp Arg Met His Leu Pro Ser Pro Ser980 985990gac tcc aag ttc tac cgc agc ctg atg agc gga gag ctg gac gag gcc 3024Asp Ser Lys Phe Tyr Arg Ser Leu Met Ser Gly Glu Leu Asp Glu Ala995 1000 1005gtg gac gca gac gag tat tta gtg ccc aat cac agc ttc ttc agc 3069Val Asp Ala Asp Glu Tyr Leu Val Pro Asn His Ser Phe Phe Ser101010151020agc ccg agc acg tcc cgc aca caa ctg ctg cac tct gtg agc ctg 3114Ser Pro Ser Thr Ser Arg Thr Gln Leu Leu His Ser Val Ser Leu102510301035aac agc agc ttt gga aac tgt aat agt aga aac ggg aat ggt tat 3159Asn Ser Ser Phe Gly Asn Cys Asn Ser Arg Asn Gly Asn Gly Tyr1040 10451050cca gtg agg gag aac agc atg gtc ctg cgc tac atc cca gac ccc 3204Pro Val Arg Glu Asn Ser Met Val Leu Arg Tyr Ile Pro Asp Pro1055 10601065aca gag cgc ttt cag gag gga gac ttt cag cct gcg ccg ggt tat 3249Thr Glu Arg Phe Gln Glu Gly Asp Phe Gln Pro Ala Pro Gly Tyr1070 10751080aac gaa tat atg aac cag aat gag tcc agc atg atc aac cca gtg 3294Asn Glu Tyr Met Asn Gln Asn Glu Ser Ser Met Ile Asn Pro Val1085 10901095tac cag cag ccc cac gga ccc ccg cgg acc ctc ctc cac tcc tcc 3339Tyr Gln Gln Pro His Gly Pro Pro Arg Thr Leu Leu His Ser Ser1100 11051110cca gcg ctg gac gag acg gaa gag gag tat ctg aac tgc ttc aag 3384Pro Ala Leu Asp Glu Thr Glu Glu Glu Tyr Leu Asn Cys Phe Lys1115 11201125agc ccg gct ccg gct tca gtg gtg gag tat ctg aac acg tcc cac 3429Ser Pro Ala Pro Ala Ser Val Val Glu Tyr Leu Asn Thr Ser His
1130 1135 1140aca cag ctg ctc tcc aca aag ccc ttc ttc agc atg gac aac ccc 3474Thr Gln Leu Leu Ser Thr Lys Pro Phe Phe Ser Met Asp Asn Pro1145 1150 1155gac tac cag cag gac ttc tgc ccg ctg gag ctc aaa aca cac acc 3519Asp Tyr Gln Gln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr1160 1165 1170aac ggg cac ctg ccg gcc gcg cag aac cag gag tac atg ggc ctg 3564Asn Gly His Leu Pro Ala Ala Gln Asn Gln Glu Tyr Met Gly Leu1175 1180 1185gag gtg cac tag 3576Glu Val His1190210272111191212PRT213斑馬魚(Danio reno)40027Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe Thr Leu Leu Leu Ser1 5 1015Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys Val Cys Gln Gly Ala Asn20 25 30Asn Lys Leu Thr Leu Leu Gly Thr Val Glu Asp His Tyr Gln Val Leu35 4045Leu Arg Met Tyr Arg Asn Cys Thr Val Val Leu Glu Asn Leu Glu Ile505560Thr His Ile Thr Glu Lys Tyr Asp Leu Ser Phe Leu Lys Ser Ile Gln65 70 7580Glu Val Gly Gly Tyr Val Leu Ile Ala Val Asn Thr Val Ser Lys Ile85 90 95Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly His Ser Leu Tyr Glu Asp100 105 110Lys Phe Ala Leu Ala Val Leu Val Asn Phe Asn Asn Ser Ile Glu Gln115 120125Gly Val Lys Glu Leu Pro Leu Thr Ser Leu Thr Glu Ile Leu Lys Gly
130135140Gly Val Lys Phe Cys Arg Asn Asp Tyr Leu Cys Asn Val Gly Thr Ile145 150 155160Glu Trp Ala Asp Ile Leu Asn Met Lys Ser Leu Pro Thr Ile Val Ser165 170175His Asn Ile Ser Tyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys180 185190Phe Asn Gly Ser Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met195200 205Thr Lys Val Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro210 215220Arg Pro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly225 230235 240Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp Glu Gly245250255Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro Asn Thr260265270His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly Ala Thr275 280 285Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp His Gly Ala290 295300Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val Asp Glu Gly Gly305310 315320Val Arg Lys Cys Lys Arg Cys Glu Gly Leu Cys Pro Lys Val Cys Asn325330335Gly Leu Gly Met Gly Pro Leu Ala Asn Val Leu Ser Ile Asn Ala Thr340 345 350Asn Ile Asp Ser Phe Glu Asn Cys Thr Lys Ile Ser Gly Asn Val Ala355360365Ile Leu Ser Thr Thr Phe Arg Gly Asp Pro His Thr Asn Thr Ser Gly370375380Leu Asp Pro Ala Lys Leu Ser Val Leu Ser Thr Val Lys Glu Ile Thr
385390 395400Gly Tyr Leu Met Ile Gln Leu Trp Pro Glu Ser Met Gln Ser Leu Ser405 410415Ala Phe Glu Asn Leu Glu Val Ile Arg Gly Arg Thr Lys Thr Gln Gly420425 430Thr Tyr Ser Phe Ala Val Thr Lys Thr Ala Ile Thr His Leu Gly Met435 440 445Arg Ser Leu Arg Glu Ile Ser Asp Gly Asp Val Ser Ile Val Lys Asn450 455 460Lys Asn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys Arg Leu Phe Lys465470475 480Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn Met Asp Ala Ala Thr485 490495Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met Cys Thr Ala Asp Gly500505510Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly Cys Glu His Tyr Ser515 520525Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu Leu Asn Gly Glu Pro530535 540Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu Cys Asp Pro Glu Cys545 550555560Leu Leu Met Asn Glu Thr Gln Thr Cys Asn Gly Pro Gly Pro Asp Lys565 570 575Cys Thr Val Cys Ala Asn Tyr Lys Asp Gly Pro His Cys Val His Arg580 585590Cys Pro Gln Gly Val Pro Gly Glu Lys Asp Thr Leu Ile Trp Lys Tyr595600 605Ala Asp Val Thr His Val Cys Gln Pro Cys His Glu Asn Cys Thr Gln610 615620Gly Cys Thr Gly Pro Asp Leu Lys Asp Cys Lys Asp Phe Lys Ser Ser625 630635 640Gly Leu Pro Met Ile Ala Ala Gly Val Val Gly Gly Leu Leu Ala Phe
645 650 655Val Ile Leu Ala Leu Gly Val Ala Val Leu Leu Arg Arg Arg His Ile660 665670Arg Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val675 680685Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg690695 700Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly705 710 715 720Ala Phe Gly Thr Val His Lys Gly Leu Trp Val Pro Glu Gly Glu Asn725 730735Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Ala Thr Ser Pro740 745750Lys Ala Asn Lys Glu Ile Met Asp Glu Ala Tyr Val Met Ala Ser Val755 760765Glu His Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr770 775780Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp Tyr785 790795 800Val Arg Glu Asn Lys Asp Arg Ile Gly Ser Gln His Leu Leu Asn Trp805810 815Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Glu Arg His Leu820 825 830Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln835840845His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Asn Ala Asp850 855860Glu Lys Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met865 870 875 880Ala Leu Glu Ser Ile Gln His Arg Thr Tyr Thr His Gln Ser Asp Val885 890895Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Thr Lys
900 905 910Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ala Gly Val Leu Glu Lys915 920 925Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met930935 940Ile Met Val Lys Cys Trp Met Ile Asp Ala Glu Ser Arg Pro Arg Phe945 950 955960Arg Glu Leu Ile Ala Glu Phe Thr Lys Met Ala Arg Asp Pro Ser Arg965 970975Tyr Leu Val Ile Gln Gly Asp Asp Arg Met His Leu Pro Ser Pro Ser980 985990Asp Ser Lys Phe Tyr Arg Ser Leu Met Ser Gly Glu Leu Asp Glu Ala99510001005Val Asp Ala Asp Glu Tyr Leu Val Pro Asn His Ser Phe Phe Ser101010151020Ser Pro Ser Thr Ser Arg Thr Gln Leu Leu His Ser Val Ser Leu102510301035Asn Ser Ser Phe Gly Asn Cys Asn Ser Arg Asn Gly Asn Gly Tyr104010451050Pro Val Arg Glu Asn Ser Met Val Leu Arg Tyr Ile Pro Asp Pro105510601065Thr Glu Arg Phe Gln Glu Gly Asp Phe Gln Pro Ala Pro Gly Tyr107010751080Asn Glu Tyr Met Asn Gln Asn Glu Ser Ser Met Ile Asn Pro Val108510901095Tyr Gln Gln Pro His Gly Pro Pro Arg Thr Leu Leu His Ser Ser110011051110Pro Ala Leu Asp Glu Thr Glu Glu Glu Tyr Leu Asn Cys Phe Lys111511201125Ser Pro Ala Pro Ala Ser Val Val Glu Tyr Leu Asn Thr Ser His113011351140Thr Gln Leu Leu Ser Thr Lys Pro Phe Phe Ser Met Asp Asn Pro
11451150 1155Asp Tyr Gln Gln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr11601165 1170Asn Gly His Leu Pro Ala Ala Gln Asn Gln Glu Tyr Met Gly Leu11751180 1185Glu Val His1190
權利要求
1.一種製備疫苗的方法，該方法包括1)分析特定病原體的特異抗原；2)獲得編碼特異抗原的多核苷酸序列；3)獲得與該多核苷酸序列有足夠差異的多核苷酸序列；4)利用步驟3)中所得的多核苷酸序列製備疫苗。
2.如權利要求1所述的方法，其中所述病原體選自細菌、病毒、真菌和原生動物。
3.如權利要求1所述的方法，其中步驟1)所述的多核苷酸序列與步驟3)所述的多核苷酸序列的差異，以它們所編碼的多肽序列而言，同源性為30％-95％。
4.如權利要求1所述的方法，其中所述疫苗的形式選自重組蛋白疫苗、重組基因疫苗、重組病毒疫苗、基因修飾疫苗及穩定轉化共生菌。
5.一種製備腫瘤疫苗的方法，該方法包括1)分析動物體內的腫瘤細胞的特異抗原；2)獲得編碼特異抗原的多核苷酸序列；3)獲得與該多核苷酸序列有足夠差異的多核苷酸序列；4)利用步驟3)中所得的多核苷酸序列製備疫苗。
6.如權利要求5所述的方法，其中所述腫瘤細胞發生在動物體內。
7.如權利要求5所述的方法，其中所述腫瘤細胞來源於動物體。
8.如權利要求6或7所述的方法，其中所述動物體包括人體。
9.如權利要求5所述的方法，其中步驟1)所述的多核苷酸序列與步驟3)所述的多核苷酸序列的差異，以它們所編碼的多肽序列而言，同源性為30％-95％。
10.如權利要求5所述的方法，其中所述疫苗的形式選自重組蛋白疫苗、重組基因疫苗、重組病毒疫苗、基因修飾疫苗及穩定轉化共生菌。
11.一種製備EGFR疫苗的方法，該方法包括1)分析動物體內的腫瘤細胞的EGFR抗原；2)獲得編碼特異抗原的多核苷酸序列；3)獲得與該多核苷酸序列有足夠差異的多核苷酸序列；4)利用步驟3)中所得的多核苷酸序列製備疫苗。
12.如權利要求11所述的方法，其中所述腫瘤細胞發生在動物體內。
13.如權利要求11所述的方法，其中所述腫瘤細胞來源於動物體。
14.如權利要求11所述的方法，其中步驟1)所述的多核苷酸序列與步驟3)所述的多核苷酸序列的差異，以它們所編碼的多肽序列而言，同源性為30％-95％。
15.如權利要求11所述的方法，其中所述疫苗的形式選自選自重組蛋白疫苗、重組基因疫苗、重組病毒疫苗、基因修飾疫苗及穩定轉化共生菌。
16.一種核酸疫苗，其中含有的多核苷酸序列所編碼的多肽與病原體中特定抗原的多核苷酸序列所編碼的多肽的同源性為30-95％。
17. 一種表皮生長因子受體(EGFR)核酸疫苗，其中含有的多核苷酸序列編碼的表皮生長因子受體與生物體內的該表皮生長因子受體中的胺基酸序列的同源性為30-95％。
18.一種表皮生長因子受體(EGFR)核酸疫苗，其中含有的多核苷酸序列編碼的表皮生長因子受體與人體內的該表皮生長因子受體中的胺基酸序列的同源性為30-95％。
19.一種製備用於治療人體腫瘤的EGFR疫苗的方法，該方法包括1)從異種生物獲得編碼EGFR的多核苷酸序列；和2)利用步驟1)中所得的多核苷酸序列製備疫苗。
20.如權利要求19所述的方法，其中所述異種生物是除人之外的其他生物，如小鼠、大鼠、雞、鮐、果蠅等。
21.如權利要求19所述的方法，其中所述從異種生物獲得編碼EGFR的多核苷酸序列選自SEQ.ID.No.13，SEQ.ID.No.15，SEQ.ID.No.17，SEQ.ID.No.19，SEQ.ID.No.21，SEQ.ID.No.23，SEQ.ID.No.24和SEQ.ID.26。
22.如權利要求19所述的方法，其中所述疫苗的形式選自選自重組蛋白疫苗、重組基因疫苗、重組病毒疫苗、基因修飾疫苗及穩定轉化共生菌。
23.如權利要求19的方法，其中人體腫瘤選自過量表達EGFR分子的肺癌、乳腺癌、卵巢癌、結腸癌、前列腺癌、胃癌、膀胱癌、頭頸部鱗癌和膠質瘤等。
24.一種製備用於治療人體腫瘤的EGFR疫苗的方法，該方法包括1)修飾人EGFR的多核苷酸序列；和2)從步驟1)中所得的多核苷酸序列中選擇修飾後的多核苷酸序列製備疫苗。
25.如權利要求24的方法，其中所述人EGFR的多核苷酸序列選自SEQ.ID.No.1，SEQ.ID.No.3，SEQ.ID.No.5，SEQ.ID.No.7，SEQ.ID.No.9和SEQ.ID.No.11。
26.如權利要求24的方法，其中所述修飾方法選自易錯PCR、改組、寡核苷酸介導的誘變、裝配PCR、有性PCR誘變、體內誘變、盒式誘變、遞歸集團誘變、指數集團誘變、位點特異性誘變、基因再裝配、GSSM及其任意組合。
27.如權利要求24所述的方法，其中所述疫苗的形式選自選自重組蛋白疫苗、重組基因疫苗、重組病毒疫苗、基因修飾疫苗及穩定轉化共生菌。
28.如權利要求24的方法，其中人體腫瘤選自過量表達EGFR分子的肺癌、乳腺癌、卵巢癌、結腸癌、前列腺癌、胃癌、膀胱癌、頭頸部鱗癌和膠質瘤等。
29.一種表皮生長因子受體多肽疫苗，其中含有的表皮生長因子受體多肽與人體內的該表皮生長因子受體中的胺基酸序列的同源性為30-95％。
全文摘要
本發明提供了一種製備疫苗的方法，包括1)分析特定病原體的特異抗原；2)獲得編碼特異抗原的多核苷酸序列；3)獲得與該多核苷酸序列有足夠差異的多核苷酸序列；4)利用步驟3)中所得的多核苷酸序列製備疫苗。本發明也提供了抗腫瘤疫苗EGFR分子疫苗，可以是常規製劑，如水劑，也可以是利用納米生物技術製備而成的納米粒製劑。EGFR分子疫苗的抗腫瘤作用還可在其它免疫促進因子的協同作用下得到增強。
文檔編號A61K39/00GK1548537SQ20031011305
公開日2004年11月24日 申請日期2003年12月25日 優先權日2002年12月27日
發明者魏於全, 田聆 申請人:深圳市清華源興生物醫藥科技有限公司