經修飾的DKK2蛋白、對其編碼的核酸、其製備方法及其用途與流程

2023-10-06 06:35:19

本公開涉及一種添加有糖基或具有改善的結合親和性的經修飾的dkk2蛋白、對其編碼的核酸、其製備方法及其用途。
背景技術：
：糖基化是糖與蛋白質連接的工藝，主要分為n-糖基化和o-糖基化。糖基化由糖基轉移酶催化，並已經報導有約200種糖基轉移酶。糖的種類和結構可能會影響蛋白質摺疊、穩定性、溶解度和對蛋白酶的敏感性、血清半衰期、抗原性、活性增加等。dkk2是wnt的阻遏蛋白，屬於dickkopf家族，並已經報導作為wnt信號通路的抑制因子或刺激因子(wuw等,curr.biol.,10(24),第1611-1614頁,2000)。dkk2可以包含兩個特定的富含半胱氨酸的結構域(crd)，並包括不同長度的連接區域。特別地，dkk2在具有10個半胱氨酸胺基酸的dickkopf家族成員中高度保守半胱氨酸-2區域(krupnikve等,gene,238(2),第301-313頁,1999)。dkk2是在動物細胞中因低表達效率而難以產生的蛋白質，因此使用dkk2的治療劑的開發正在延遲。因此，需要能夠維持結合親和性或對其底物具有改善的結合親和性並且還表現出增加的表達效率的dkk2。技術實現要素：問題的解決方案一方面，提供經修飾的dkk2多肽，其與野生型dkk2多肽的胺基酸序列相比，包含一個或多個額外糖基化位點。另一方面，提供編碼經修飾的dkk2多肽的核酸。又另一方面，提供製備經修飾的dkk2多肽的方法。又另一方面，提供用於促進血管生成的藥物組合物，所述組合物包含經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸、以及藥學上可接受的載體。又另一方面，提供用於預防或治療血管通透性相關疾病的藥物組合物，所述組合物包括經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸、以及藥學上可接受的載體。又另一方面，提供一種促進受試者的血管生成的方法，所述方法包括將經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸施用於所述受試者。又另一方面，提供一種預防或治療受試者的血管通透性相關疾病的方法，所述方法包括將經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸施用於受試者。發明的有益效果根據一方面的經修飾的dkk2多肽、對其編碼的核酸、其製備方法及其用途，可以有效製備具有額外糖基或對其底物lrp6具有改善的結合親和性的經修飾的dkk2蛋白，從而被用於促進血管生成或用於預防或治療血管通透性相關疾病。附圖說明從以下結合附圖對示例性實施方式的描述中，這些和/或其它方面將變得顯而易見和更容易理解，其中：圖1a和圖1b分別是示出了含有各種標籤的野生型dkk2蛋白和突變型dkk2蛋白的免疫印跡結果的圖像，圖1c是示出了含有fc標籤的dkk2n-gly4和dkk2n-gly5蛋白的免疫印跡結果的圖像，圖1d是示出了純化蛋白質的電泳結果的圖像。圖2a和圖2b是示出了突變型dkk2蛋白對各200ng的mlrp6和hlrp6在490nm處的吸光度的圖，圖2c和圖2d是示出了dkk2蛋白對各100ng的mlrp6和hlrp6在490nm處的吸光度的圖；圖3a和圖3b是示出了在施用試驗材料後第14天和第21天施用突變型dkk2蛋白質的視網膜的螢光強度(％)的圖；以及圖4是示出了伊文思藍血管通透性(％)根據施用dkk2蛋白質的圖。具體實施方式一方面，提供了與野生型dickkopf(dkk)2多肽的胺基酸序列相比包含一個或多個額外糖基化位點的經修飾的dkk2多肽。dkk2是指dickkopf-2蛋白，也稱為dickkopf相關蛋白2、富含半胱氨酸的分泌蛋白2、crsp2、crispy2或crsp2蛋白。dkk2是一種在人類中由dkk2基因編碼的蛋白質。該基因編碼作為dickkopf家族成員的蛋白質。已知dkk2是一種分泌蛋白質，含有兩個富含半胱氨酸的區域，通過與wnt信號通路的相互作用而參與胚胎發育。此外，它可以作為wnt/β-連環蛋白信號傳導的激動劑或拮抗劑，這取決於細胞情況和輔因子kremen2的存在。dkk2可以與低密度脂蛋白受體相關蛋白6(lrp6)結合。野生型dkk2多肽可以是野生型dkk2前體多肽。野生型dkk2前體多肽可以是人類中具有基因庫登錄號為np_055236.1的胺基酸序列(序列號1)的多肽或小鼠中具有基因庫登錄號為np_064661.2的胺基酸序列的多肽。野生型dkk2前體多肽可以包括信號肽，並且信號肽可以通過共翻譯修飾或翻譯後修飾來切割。野生型dkk2多肽可以是成熟dkk2。成熟dkk2可以是包括通過從序列號1的胺基酸序列的n-末端去除位置1至33處的胺基酸序列(信號肽或前導肽)而得到的胺基酸序列的多肽，或包括序列號3的胺基酸序列的多肽。野生型dkk2前體多肽可以通過人類中具有基因庫登錄號為nm_014421的核苷酸序列(序列號4)的核酸以及小鼠中具有基因庫登錄號為nm_020265的核苷酸序列的核酸來編碼。糖基化是指將糖基轉移到蛋白質的反應。糖基化由糖基轉移酶催化。糖基化可以是n-糖基化、o-糖基化、磷酸-絲氨酸糖基化、c-甘露糖基化、糖基磷脂醯肌醇化或其組合。n-糖基化是指糖分子與天冬醯胺的醯胺基團的連接。糖基化位點是指可以連接有糖分子或糖基的位點。例如，糖基化位點可以是asn-xaa-ser/thr的共有序列中的天冬醯胺殘基(其是n-糖基化位點)。asn表示天冬醯胺，xaa表示除脯氨酸以外的胺基酸，ser表示絲氨酸，thr表示蘇氨酸，ser/thr表示絲氨酸或蘇氨酸。asn-xaa-ser/thr表示從n-末端起由天冬醯胺-胺基酸(不包括脯氨酸)-絲氨酸或胺基酸組成的多肽。糖分子與asn-xaa-ser/thr的天冬醯胺連接。野生型dkk2前體多肽可以從n-末端在位置36處包括一個糖基化位點。經修飾的dkk2多肽的糖基化位點可以通過將天門冬醯胺(asn，n)取代序列號3的胺基酸序列中選自由5i、31g、96p、110d、44p、2l、45c、57c、62q、63g、85p、6k、98t、101i、11g、121h、135p、138k、151l、152r、166f、187k、203g、211d、213t和214y組成的組中的一種或多種胺基酸而引入。數字表示從序列號3的胺基酸序列的n-末端起胺基酸所在的位置，並且字母字符表示胺基酸的1個字母代碼。例如，『5i』表示從序列號3的胺基酸序列的n-末端起位置5處的異亮氨酸。修飾可以是一個或多個胺基酸的取代。經修飾的dkk2多肽可以是dkk2多肽，其中通過引入糖基化位點而額外引入糖基。儘管經修飾的dkk2多肽可以在糖鍵、糖組成、糖基結構或其組合中變化，但糖基化發生在dkk2多肽的胺基酸序列的相同糖基化位點。作為結果，可以增加細胞內表達效率。例如，雖然經修飾的dkk2多肽可以在糖鍵(例如n-糖基化和o-糖基化)、糖組成(例如唾液酸化程度的差異)和糖基結構中根據宿主細胞(其中經修飾的dkk2多肽被表達)的種類而變化，但糖基化可以發生在dkk2多肽的相同糖基化位點。經修飾的dkk2多肽可以是包含一個或多個選自序列號5至30的胺基酸序列的多肽。標籤可以進一步包含在經修飾的dkk2多肽的n-末端或c-末端。標籤可以是連接到dkk2多肽的多肽，以促進表達、純化、檢測等。標籤可以是例如fc(片段可結晶)區、多組氨酸肽或其組合。fc區可以是人fc區、小鼠fc區等。fc區可以是由序列號48的核苷酸序列編碼的多肽。fc區可以是由序列號49的核苷酸序列組成的多肽。標籤可以是本領域技術人員已知的。經修飾的dkk2多肽可以進一步包括n-末端處的信號肽。信號肽是指存在於大部分新合成的蛋白質的n-末端的肽(大約5-30個胺基酸長)，其註定分泌途徑。信號肽可以是序列號1的胺基酸序列的n-末端起位置1至33處的胺基酸序列或包含序列號2的胺基酸序列的多肽。與野生型dkk2多肽相比，經修飾的dkk2多肽可以是具有額外糖基、lrp6結合親和性的增加、或其組合的多肽。與作為對照組的野生型dkk2多肽相比，在經修飾的dkk2多肽中的額外糖基可以是糖基量的增加。由於額外糖基，與野生型dkk2多肽的量相比，經修飾的dkk2多肽的量可能在細胞中增加。與編碼野生型dkk2多肽的轉錄物相比，編碼經修飾的dkk2多肽的轉錄物的量可以在細胞中增加。多肽或轉錄物的量的增加可能意味著多肽的細胞內表達效率增加。與野生型dkk2多肽相比，經修飾的dkk2多肽對lrp6的結合親和性可能增加。結合親和性越高，解離常數(kd)可能越低。經修飾的dkk2多肽對於lrp6的解離常數可以是例如約0.1nm至約100nm、約1nm至約50nm、約2nm至約40nm、約3nm至約30nm、約4nm至約20nm、約5nm至約10nm、或約5.5nm。另一方面，提供一種編碼經修飾的dkk2多肽的核酸。該經修飾的dkk2多肽與上述相同。核酸可以包括選自序列號43至47中的任一個核苷酸序列。核酸在編碼經修飾的dkk2多肽的核酸的5'-末端或3'-末端處還可以包括編碼標籤的核苷酸序列。核酸在編碼經修飾的dkk2多肽的核酸的5'-末端處還可以包括編碼信號肽的核苷酸序列。核酸可以可操作地連接到基因表達調節元件，例如啟動子、操縱子、增強子和/或轉錄終止子。另一方面，提供一種製備經修飾的dkk2多肽的方法，所述方法包括：在培養基的存在下培養用包含編碼經修飾的dkk2多肽的核酸的載體引入的細胞，以獲得培養產物；並從培養產物獲得經修飾的dkk2多肽。該經修飾的dkk2多肽和編碼經修飾的dkk2多肽的核酸與上述相同。細胞可以是真核細胞。例如，細胞可以是動物細胞。細胞可以是例如胚胎腎細胞、卵巢細胞、骨髓瘤細胞或視網膜衍生細胞。胚胎腎細胞可以是人胚胎腎(hek)293細胞或幼倉鼠腎(bhk)細胞。卵巢細胞可能是中國倉鼠卵巢細胞(cho)細胞。骨髓瘤細胞可以是ns0細胞或sp2/0細胞。視網膜衍生細胞可以是perc6細胞。根據細胞的種類，在糖鍵(例如n-糖基化和o-糖基化)、糖組成(例如唾液酸化程度的差異)和糖基結構中變化的dkk2多肽可以被表達。該方法還可以包括將包含編碼經修飾的dkk2多肽的核酸的載體引入細胞。載體可以是例如質粒、病毒載體、粘粒或人造染色體。載體可以是包含啟動子序列的表達載體。載體可以是能夠在真核細胞中表達靶基因的載體。引入是指將核酸引入細胞。引入可以是例如通過轉化、突變、轉導、綴合或電穿孔引入。該方法包括在培養基的存在下培養引入的細胞以獲得培養產物。培養基是指含有促進細胞存活或增殖所必需或有作用的成分的培養基。根據細胞種類，本領域技術人員可以選擇培養基。可以使用本領域技術人員已知的方法進行孵育來進行培養。培養可以在例如約37℃的溫度和5％co2條件下進行。該方法包括從培養產物得到經修飾的dkk2多肽。培養產物可以是不包括培養細胞的培養液或培養細胞。得到經修飾的dkk2多肽可以包括得到和裂解細胞，並純化或過濾多肽。得到多肽的方法可以是本領域技術人員已知的。另一方面，提供一種用於促進血管生成的藥物組合物，所述組合物包含經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸、以及藥學上可接受的載體。該經修飾的dkk2多肽和編碼經修飾的dkk2多肽的核酸與上述相同。血管生成是指形成新血管的過程。血管生成包括新血管從預先存在的血管生長的過程。血管生成是傷口癒合和肉芽組織以及生長和發育中的正常和重要的過程。此外，血管生成是腫瘤從休眠狀態轉變為惡性狀態的基本步驟。促進可以在體外或體內實現。促進可能誘導具有缺血性血管疾病的受試者的新血管的形成或再生。藥物組合物可以是用於預防或治療缺血性血管疾病的藥物組合物。缺血性血管疾病可以是例如燒傷、牛皮癬、潰瘍、缺血、缺血性心臟病、缺血性腦血管疾病或勃起功能障礙。另一方面，提供一種用於預防或治療血管通透性相關疾病的藥物組合物，所述組合物包含經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸、以及藥學上可接受的載體。該經修飾的dkk2多肽和編碼經修飾的dkk2多肽的核酸與上述相同。血管通透性相關疾病是指具有由於血管通透性增加而導致身體流體進入周圍組織的洩漏增加而引起的症狀的疾病。血管通透性相關疾病可以是例如糖尿病性視網膜病變、糖尿病性黃斑水腫、視網膜靜脈閉塞後的黃斑水腫、黃斑變性(例如新生血管(溼性)年齡相關性黃斑變性)、脈絡膜新生血管形成、青光眼視網膜色素變性、視網膜病變早產兒(rop)、青光眼、角膜營養不良、視網膜疾病、斯塔格特氏病、常染色體顯性藥物、最佳黃斑營養不良、囊性黃斑水腫、缺血性視網膜病變、炎症誘導的視網膜退行性疾病、x連鎖青少年視網膜病變、malattialeventinese(ml)或doyne蜂窩狀視網膜營養不良症。如本文所用，術語「預防」是指通過施用藥物組合物來抑制或延緩疾病發生的所有作用。術語「治療」是指通過施用藥物組合物有利於症狀好轉或改善的所有作用。藥物組合物可以包括藥學上可接受的載體。載體包括賦形劑、稀釋劑或輔助物質。載體可以是例如選自乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖醇、木糖醇、赤蘚糖醇、麥芽糖醇、澱粉、金合歡膠、藻酸鹽、明膠、磷酸鈣、矽酸鈣、纖維素、甲基纖維素、聚乙烯基吡咯烷酮、水、生理鹽水溶液、緩衝液例如pbs、羥基苯甲酸甲酯、羥基苯甲酸丙酯、滑石、硬脂酸鎂和礦物油。組合物可以包括填料、抗凝集劑、潤滑劑、潤溼劑、風味劑、乳化劑、防腐劑等。藥物組合物可以以任何製劑製備。組合物可以配製成口服劑型(例如粉劑、片劑、膠囊劑、糖漿劑、丸劑、顆粒劑)或腸胃外劑型(例如可注射製劑)。此外，組合物可以以全身劑型或局部劑型製備。藥物組合物可以包括有效量的經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸。有效量可以根據本領域技術人員選擇的細胞或受試者適當地選擇。有效量可以根據疾病的嚴重程度、患者的年齡、體重、健康狀況、性別和藥物敏感性、給藥時間、給藥途徑、排洩率、治療期、和與組合物混合或共同給藥的藥物以及醫療領域中眾所周知的其它因素來確定。基於藥物組合物，有效量可以為約0.5μg至約2g、約1μg至約1g、約10μg至約500mg、約100μg至約100mg、約1mg至約50mg。藥物組合物的給藥劑量可以是例如每個成人約0.001mg/kg至約100mg/kg、約0.001mg/kg至約10mg/kg、約0.001mg/kg至約1mg/kg、約0.005mg/kg至約1mg/kg、約0.01mg/kg至約1mg/kg、或約0.1mg/kg至約1mg/kg，每天一次、每天數次、每周兩次或三次、每月一次至四次、每年一至十二次。另一方面，提供一種促進受試者的血管生成的方法，所述方法包括將經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸施用於受試者。另一方面，提供一種預防或治療受試者的血管通透性相關疾病的方法，所述方法包括將經修飾的dkk2多肽或編碼經修飾的dkk2多肽的核酸施用於受試者。該經修飾的dkk2多肽、編碼經修飾的dkk2多肽的核酸、血管生成、促進、血管通透性相關疾病、預防和治療與上述相同。受試者可以是哺乳動物，例如人、牛、馬、豬、狗、綿羊、山羊或貓。受試者可能是具有缺血性疾病或高可能性具有缺血性疾病的受試者。受試者可以是具有血管通透性相關疾病或高可能性具有血管通透性相關疾病的受試者。可以通過任何方式直接對受試者進行給藥，例如口服、靜脈內、肌肉內、透皮、黏膜、鼻內、氣管內或皮下給藥。給藥可以是局部或全身給藥。現在將詳細參考示例性實施方式，其示例在附圖中示出，其中相同的附圖標記始終表示相同的元件。在這方面，本示例性實施方式可以具有不同的形式，並且不應被解釋為限於本文所闡述的描述。因此，下面僅通過參考附圖描述示例性實施方式來解釋各方面。如本文所用，術語「和/或」包括一個或多個相關列出的項目的任何和所有組合。以下參照實施例對本發明進行更詳細的說明。然而，這些實施例僅用於說明的目的，本發明的範圍並不受這些實施例的限制。實施例1.n-糖基化突變型dkk2蛋白的製備和鑑定1.dkk2蛋白的n-糖基化位點的預測野生型dkk2蛋白的胺基酸序列(序列號3)從n-末端起在位置3處具有n-糖基化位點。為了人工誘導野生型dkk2蛋白中的n-糖基化，使用netnglyc1.0伺服器(www.cbs.dtu.dk/services/netnglyc/)預測野生型dkk2蛋白的n-糖基化位點。從netnglyc1.0伺服器預測了共26種dkk2蛋白的n-糖基化位點，結果如表1中所示。在表1中，胺基酸代表野生型dkk2蛋白的胺基酸(序列號3)和由其突變的天冬醯胺(asn，n)，數字表示從野生型dkk2蛋白的n-末端起突變胺基酸所在的位置。[表1]在26種dkk2蛋白的n-糖基化位點候選中，選擇了在netnglyc1.0伺服器中顯示高n-糖基化潛能的5種突變型dkk2蛋白，並檢測了蛋白質生產效率。所選擇的5種突變型dkk2蛋白的n-糖基化潛能在下表2中給出。[表2]突變型dkk2蛋白n-糖基化的潛能dkk2n-gly10.7342dkk2n-gly20.7206dkk2n-gly30.7010dkk2n-gly40.7541dkk2n-gly50.73722.所選擇的n-糖基化dkk2蛋白的生產效率的驗證為了表達實施例1.1中選擇的5種dkk2蛋白質，將其各自插入高效表達載體中，以通過瞬時表達系統檢查蛋白質生產效率。(1)聚合酶鏈反應使用野生型dkk2天然形式(基因庫登錄號np_055236.1，序列號3)(由medpacto,inc.,korea提供)作為模板。設計用於dkk2n-糖基化的引物的核苷酸序列在表3中給出。[表3]對於聚合酶鏈反應，製備以下組成的混合物。100ng模板2.5單位pfudna聚合酶(spx16-rs500,solgentco.,ltd.)10pmol正向引物10pmol反向引物1μl10mmdntp5μl10xpfu聚合酶緩衝液50μl總體積將製備的混合物在95℃下孵育2分鐘，然後重複30次循環，每次循環包括95℃下20秒、64℃下40秒、72℃下1分鐘，然後在72℃下孵育10分鐘。因此，得到如此擴增的突變型dkk2核酸。(2)擴增產物的克隆和宿主細胞中瞬時蛋白表達的檢測將擴增的突變型dkk2核酸或野生型dkk2核酸在10單位的限制酶sfii(目錄號r033s，enzynomics，korea)和1x反應緩衝液的存在下在50℃下孵育6小時。將反應產物在瓊脂糖凝膠上電泳，使用凝膠純化試劑盒(目錄號1014876，qiagen，usa)純化插入載體的核酸。將哺乳動物表達載體n293f-fc(atp-100，anrt)和純化的核酸以1:3的重量比混合，隨後在10單位的t4dna連接酶(目錄號m001s，enzynomics，korea)和1x反應緩衝液的存在下在22℃下孵育4小時以上。將反應產物轉化到大腸桿菌中，通過測序分析檢查編碼dkk2n-gly1、dkk2n-gly2、dkk2n-gly3、dkk2n-gly4或dkk2n-gly5(分別為序列號43至47)的核苷酸序列。選擇包含引入有突變型dkk2核酸的n293f-fc載體的克隆。從選擇的克隆得到引入有突變型dkk2核酸(mdkk2)的n293f-fc-mdkk2載體。fc核酸是編碼人免疫球蛋白g1的fc片段的核酸(序列號48)。將哺乳動物hek293f懸浮細胞以5×105個細胞/ml的密度接種於自由式培養基(目錄號1508027，invitrogen，usa)中，並在37℃和5％co2的條件下培養。培養細胞直到細胞的密度達到約1×106細胞/ml(約24小時)。將25μg引入有突變型dkk2核酸的n293f-fc載體、50μg聚乙烯亞胺(pei)(目錄號23966，polysciences，usa)和600μlpmi培養基(目錄號sh30027.01，hiclone，usa)混合，並在室溫下孵育20分鐘。向培養的hek293f細胞中加入反應產物，並在37℃和5％co2的條件下培養約5天。然後，為了得到由細胞分泌的水溶性蛋白質，從培養產物中除去細胞，僅收集不包括細胞的培養液。將200μl培養液進行10％sds-page，然後進行免疫印跡。由於得到的突變型dkk2蛋白和野生型dkk2蛋白在其n-末端包含fc-標籤、his-標籤或mfc-標籤，使用以1：4000稀釋的抗fc-hrp(目錄號31414，pierce，usa)、以1：2000稀釋的抗his-hrp(目錄號a7058，sigma，usa)或以1：2000稀釋的抗mfc(目錄號31430，pierce，usa)。作為顯色試劑，使用eclkit(目錄號0034077geusa)以得到圖像。圖1a示出了含有各種標籤的野生型dkk2蛋白的免疫印跡結果(暴露時間：1分鐘，m：標記物(kda)，1：n-fc-dkk2，2：n-fc(igg4)-dkk2，3：n-his-dkk2，4：n-mfc-dkk2)。如圖1a所示，即使暴露1分鐘，也沒有檢測到野生型dkk2蛋白。因此，證實了野生型dkk2蛋白幾乎不表達。圖1b示出了包含各種標籤的突變型dkk2蛋白的免疫印跡結果(暴露時間：1分鐘(左)或1秒(右)，m：標記(kda)，1：n-mfc-dkk2-gly1，2：n-mfc-dkk2-gly2，3：n-mfc-dkk2-gly3，4：n-fc4-dkk2-gly1，5：n-his-dkk2-gly1，6：n-fc-dkk2-gly1，7：n-his-dkk2-gly2，8：n-fc4-dkk2-gly2，9：n-fc-dkk2-gly2，10：n-fc-dkk2-gly3，11：n-his-dkk2-gly3，12：n-fc4-dkk2-gly3)。如圖1b所示，檢測到包含fc標籤的突變型dkk2蛋白，而幾乎沒有檢測到包含fc以外的標籤的突變型dkk2蛋白。因此，證實了含有fc標籤的突變型dkk2蛋白顯示出提高的表達效率。使用一次性開放柱純化表達的蛋白質，並測量其濃度。由於含有fc標籤的蛋白質顯示出提高的表達效率，因此獲得含有fc標籤的dkk2n-gly4和dkk2n-gly5蛋白。將獲得的蛋白質進行免疫印跡，結果示於圖1c(曝光時間：1秒(左)或30秒(右)，m：標記(kda)，1：n-fc-dkk2-gly4，2：n-fc-dkk2-gly5)。如圖1c所示，證實了n-fc-dkk2-n-gly4比n-fc-dkk2-n-gly5顯示出更高的表達效率。(3)蛋白質的大量生產和純化如實施例1(2)中所述，選擇用含有fc標籤和突變性dkk2核酸的載體轉化的細胞。將所選擇的細胞在37℃和5％co2的條件下培養約5天。將除細胞外的培養液在室溫下以4800rpm的速度離心20分鐘以得到上清液。使用0.22μm過濾器(目錄號pr02890，millipore，usa)進行過濾。製備裝有蛋白a珠(目錄號17-1279-03，gehealthcare，sweden)的5ml柱，並在4℃下將濾液以0.9ml/min的速率施加到珠子整夜。用100ml以上的pbs(磷酸緩衝鹽水)(目錄號71111，gibco，usa)洗滌珠子。此後，將0.1m甘氨酸-hcl(目錄號g7126，sigma，usa)施加到珠子以洗脫6個級分。加入1mtris(目錄號t-1503-5kg，sigma，usa)(ph9.0)以中和餾分。定量級分中的蛋白質，並收集含有蛋白質的級分。將級分施加到超濾離心管(目錄號ufc805024，millipore，usa)，並根據製造商的說明書離心。將1xpbs加入濃縮物中並重複離心三次，以用pbs替代作為儲存緩衝液。定量純化的蛋白質。在40ml培養液中n-fc-dkk2-gly1的量為130μg，在40ml培養液中n-fc-dkk2-gly2的量為860μg，在40ml培養液中n-fc-dkk2-gly3的量為150μg，在40ml培養液中n-fc-dkk2-gly4的量為462μg，在40ml培養液中n-fc-dkk2-gly5的量為27μg。在引入有n-糖基化位點的突變型dkk2蛋白中，n-fc-dkk2-gly2顯示出最高的表達效率，n-fc-dkk2-gly4顯示出接著最高的表達效率。因此，與野生型dkk2蛋白相比，引入了n-糖基化位點的突變型dkk2蛋白質高度表達，n-fc-dkk2-gly2和n-fc-dkk2-gly4的表達效率比野生型dkk2分別高約80倍和約50倍。為了檢查純化的蛋白質的純度，將3μg蛋白質通過10％sds-page電泳，結果示於圖1d(m：標記物(kda)，1：n-fc-dkk2-gly1，2：n-fc-dkk2-gly2，3：n-fc-dkk2-gly3，4：n-fc-dkk2-gly4，5：n-fc-dkk2-gly5)。如圖1d中所示，發現n-fc-dkk2-gly2和n-fc-dkk2-gly4的純度相似。3.突變型dkk2蛋白的結合親和性的檢測由於已知dkk2蛋白與小鼠lrp6(mlrp6)和人lrp6(hlrp6)結合，因此檢查了野生型dkk2和突變型dkk2蛋白對lrp6的結合親和性。具體地，將200nghlrp6蛋白(目錄號1505-lr-025，r&d，usa)或mlrp6蛋白(目錄號2960-lr-025，r&d，usa)應用於elisa板(登錄號439454，nunc，denmark)的每個孔，並在4℃下孵育過夜以用該蛋白質包覆該板。將200μl4％(w/v)脫脂乳(cat.no.232100，difco，france)/1xpbs施加到elisa板的孔中，並在室溫下孵育約1小時以進行封閉。此後，從elisa板中除去封閉溶液。將每個純化的dkk2蛋白和100μl1％(w/v)脫脂乳/1xpbs混合以製備100nm的dkk2蛋白，並將100nm的dkk2蛋白進行1/4系列稀釋。將稀釋的蛋白質施加到製備的elisa板上，並在室溫下孵育約2小時。此後，將板用200μlpbst洗滌5次。將2μl抗人fc-hrp(目錄號31413，thermo，usa)抗體與4ml含有1％(w/v)脫脂乳的pbs混合，並向elisa板的每個孔中加入200μl該混合物，並在室溫下孵育1小時。然後，除去elisa板的二次抗體，並用200μlpbs將該板洗滌5次。將10μlh2o2溶液(目錄號h1009-100ml，sigma，usa)、10mlpc緩衝液(5.1g檸檬酸一水合物，7.3g磷酸鈉/l(ph5.0))和一片opd(目錄號p8787-100tab，sigma，usa)混合以製備混合物，並將總體積為100μl的混合物加入到每個孔中。在室溫下在黑暗中進行孵育10分鐘，隨後進行顯色。此後，向每個孔中加入50μl終止緩衝液以終止顯色。使用elisa讀數器測量490nm處的吸光度，並根據測量的吸光度計算解離常數kd值(m)。圖2a示出了突變型dkk2蛋白對200ngmlrp6在490nm處的吸光度，圖2b示出了突變型dkk2蛋白對200nghlrp6在490nm處的吸光度(■：n-fc-dkk2-gly1，▲：n-fc-dkk2-gly2，▼：n-fc-dkk2-gly3，◆：n-fc-dkk2-gly4，●：n-fc-dkk2-gly5)。如圖2a和2b中所示，n-fc-dkk2-gly4顯示出對mlrp6和hlrp6的最大改善的結合親和性。證實了n-fc-dkk2-gly2顯示出最高的表達效率，但其對mlrp6和hlrp6的結合親和性不高。因此，n-fc-dkk2-gly4被證實是表現出改善的表達效率和對mlrp6和hlrp6的高結合親和性的突變型dkk2蛋白。為了比較在n-fc-dkk2-gly4與野生型dkk2(目錄號6628-dk-010，r&d，usa)之間的對mlrp6和hlrp6的結合親和性，以與上述相同的方式進行elisa，不同之處在於使用了100ng的lrp6。圖2c示出了dkk2蛋白對100ngmlrp6在490nm處的吸光度，圖2d示出了dkk2蛋白對100nghlrp6在490nm處的吸光度(■：n-fc-dkk2-gly4，▲：野生型dkk2)。從測量的吸光度計算出的解離常數(kd)和r2值在表4中給出。[表4]證實了n-fc-dkk2-gly4對hlrp6和mlrp6的結合親和性比野生型dkk2高約5倍至約10倍。4.通過突變型dkk2蛋白的血管生成的檢測為了檢查突變型dkk2蛋白是否能夠誘導血管生成，進行角膜袋檢測(cpa)。c57bl6小鼠(雄性，9周齡，重21.81g-23.81g)購自orientbioinc.(korea)，然後在進行實驗的動物設施中馴化約7天。為了向角膜施用藥物，製備藥物顆粒。具體地，將10g蔗糖八硫酸鹽-鋁絡合物(s0652，sigma-aldrich)溶於100ml的pbs(磷酸鹽緩衝液鹽水)中以製備10％(w/v)硫糖鋁溶液。將12g聚甲基丙烯酸2-羥基乙酯(p3932，sigma-aldrich)溶於100ml無水乙醇中以製備12％(w/v)聚hema溶液。將石蠟膜置於培養皿中並在紫外線下放置約15分鐘。將5μl12％(w/v)聚hema溶液、1μl10％(w/v)硫糖鋁溶液和4μl藥物混合。將各0.2μl該混合物分配在石蠟膜上，並將分配的顆粒在室溫下乾燥約1-2小時。將乾燥的顆粒在使用前儲存在冰箱中。將適應的小鼠(雄性，10周齡，體重2123.43g-26.11g)分為2組(每組n＝5)，並通過腹膜內注射氯胺酮和賽拉嗪(rompuntm)，拜耳公司，德國)的4:1(v/v)混合物使其麻醉。大約10分鐘後，將0.5％的普拉卡因滴入其角膜。用vongraefe白內障刀，在顯微鏡下在角膜上製成一個微口袋。將製備的顆粒植入角膜微口袋(第1天)。為了防止感染，將土黴素眼藥膏施用於眼睛。作為測試材料，使用n-fc-dkk2-gly4，每隻小鼠施用475ng的n-fc-dkk2-gly4。作為陰性對照組，使用pbs。此後，在顯微鏡下檢查角膜，通過圖像分析儀觀察血管生成和結構特徵。在第5天，在陰性對照組中未發現血管生成，而在n-fc-dkk2-gly4處理組中檢測到血管生成。計算n-fc-dkk2-gly4處理組中血管生成的面積(mm2)，結果在表5中給出。[表5]序號第1天第3天第5天第7天10.000.000.000.0020.000.000.000.0030.000.000.000.0040.000.000.020.0350.000.000.060.13如表5中所示，與陰性對照組相比，在n-fc-dkk2-gly4處理組中觀察到血管生成。5.突變型dkk2蛋白的糖尿病性視網膜病變改善作用檢查突變型dkk2蛋白是否能夠改善糖尿病性視網膜病變的症狀。慄鼠兔(雄性，10-11周齡，體重2.0kg-2.5kg)購自dreambioinc.(韓國)，然後在進行實驗的動物設施中馴化約7天。將作為糖尿病誘導劑的四氧嘧啶一水合物(sigma-aldrichco.)施用於適應的兔子的耳靜脈。在給藥後第7天(第0天)測量血糖水平，選擇具有血糖水平為300mg/dl的動物。將選擇的兔子隨機分配，使得每組的平均血糖水平平均分布。通過靜脈注射zoletil50(virbac，法國)和賽拉嗪(rompuntm，bayerag，germany)使糖尿病誘導的兔子麻醉。作為測試材料，在第0天(施用四氧嘧啶後第7天)和第7天通過玻璃體內注射施用n-fc-dkk2-gly4(pbs中)，以總共10μl/眼的量進行兩次給藥，每天一次。作為正常對照組，注射pbs而不是四氧嘧啶。作為糖尿病對照組，施用四氧嘧啶，作為陽性對照組，在第0天以50μl/眼的量通過玻璃體內注射施用(bayerkorealtd.)一次。施用藥物在下表6中給出。[表6]組動物數量施用材料一次劑量或施用頻率備註17pbs0未處理27pbs0糖尿病誘導37eylea2000μg，一次糖尿病誘導47n-fc-dkk2-gly47μg，兩次糖尿病誘導57n-fc-dkk2-gly420μg，兩次糖尿病誘導67n-fc-dkk2-gly470μg，兩次糖尿病誘導在第0天(測試材料給藥開始的那天)、第14天和第21天，將散瞳劑(mydriacyl滴眼劑1％)滴入兔眼中，並用zoletil50(法國virbac)和賽拉嗪((rompuntm)，拜耳公司，德國)麻醉。此後，通過耳靜脈注射2％(w/v)螢光素鈉鹽溶液(sigmaaldrich)，並使用眼底照相機(trc-50ix，topcon，japan)拍攝兔子的眼睛約2分鐘或更短。通過螢光眼底圖像評估視網膜和功效的檢查。使用imagej軟體(nih，bethesda，md)進行圖像分析，以測量視網膜非血管區域的螢光強度，並基於組1(正常對照組)的平均值(100％)，計算每個受試者的測量值的相對水平(％)。計算出的螢光強度示於圖3a和3b(圖3a：第14天，圖3b：第21天；*和***：與組1相比，分別為p<0.05和p<0.001；#、##和###：與組2相比，分別為p<0.05，p<0.01和p<0.001；$：與組3相比，p<0.05)。如圖3a和3b中所示，n-fc-dkk2-gly4處理組根據給藥劑量在視網膜的非血管區域中顯示出顯著低的螢光強度，表明n-fc-dkk2-gly4具有抑制視網膜非血管區域的血管滲漏的作用。此外，在第21天，用zoletil50和賽拉嗪麻醉兔子。通過耳靜脈注射1％(v/v)伊文思藍溶液(abcam)。約10分鐘後，將兔子的眼睛切下並固定在10％(v/v)中性緩衝福馬林溶液中。大約24小時後，將視網膜與眼睛分開，並將1ml蒸餾水施加到分離的視網膜上，然後渦旋約10分鐘。將混合物在10000×g和室溫下離心約10分鐘。將0.3ml上清液轉移到96孔微量培養板中，並使用versamax酶標儀(moleculardevice，usa)測量620nm處的吸光度。基於組1(正常對照組)的平均值(100％)，計算每個受試者的伊文思藍血管通透性。計算的通透性示於圖4(*和***：與組1相比，分別為p<0.05和p<0.001；##和###：與組2相比，分別為p<0.01和p<0.001；$$：與組3相比，p<0.01)。如圖4中所示，n-fc-dkk2-gly4處理組根據給藥劑量顯示出顯著低的血管通透性，表明n-fc-dkk2-gly4具有抑制視網膜血管通透性的作用。糖尿病性視網膜病變的特徵在於視網膜血管生成(或新生血管形成)、玻璃體出血等。由於n-fc-dkk2-gly4抑制血管生成、玻璃體出血和視網膜血管通透性，證實了經修飾的dkk2多肽例如n-fc-dkk2-gly4對糖尿病性視網膜病變具有預防或治療作用。應當理解，本文描述的示例性實施方式應僅在描述性意義上被考慮，而不是為了限制的目的。在其它示例性實施方式中，每個示例性實施方式中的特徵或方面的描述通常被認為可用於其它類似特徵或方面。雖然已經參考附圖描述了一個或多個示例性實施方式，但是本領域普通技術人員將會理解，在不脫離由如下權利要求所定義的精神和範圍的情況下，可以在形式和細節上進行各種改變。株式會社麥迪帕克特經修飾的dkk2蛋白、對其編碼的核酸、其製備方法及其用途px051158-pct49kopatentin2.01259prt人工序列人野生型dkk2前體1metalaalaleumetargserlysaspsersercyscysleuleuleu151015leualaalavalleumetvalgluserserglnileglyserserarg202530alalysleuasnserilelysserserleuglyglygluthrprogly354045glnalaalaasnargseralaglymettyrglnglyleualaphegly505560glyserlyslysglylysasnleuglyglnalatyrprocysserser65707580asplysglucysgluvalglyargtyrcyshisserprohisglngly859095serseralacysmetvalcysargarglyslyslysargcyshisarg100105110aspglymetcyscysproserth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