蠟狀芽孢桿菌產生的嘔吐毒素的合成酶、編碼該酶的基因以及嘔吐毒素的檢測方法

2023-09-20 08:17:55

專利名稱：蠟狀芽孢桿菌產生的嘔吐毒素的合成酶、編碼該酶的基因以及嘔吐毒素的檢測方法
技術領域：
本發明涉及蠟狀芽孢桿菌(Bacillus cereus)產生的嘔吐毒素(催吐素)及其檢測方法。在臨床檢查或食品檢查等中的催吐素的檢測中可以利用本發明。
背景技術：
人們都知道混入食品成為食物中毒原因的細菌毒素中黃色葡萄球菌瀉肚毒素和蠟狀芽孢桿菌嘔吐毒素作為耐熱性毒素，進行加熱處理是無效的。關於在食品衛生上極其重要的黃色葡萄球菌毒素已經確立了檢測方法，但關於蠟狀芽孢桿菌嘔吐毒素的檢測到目前為止還沒有開發出適當的方法。蠟狀芽孢桿菌由於形成能夠耐受在100℃下加熱30分鐘的耐熱性芽孢，通過煮沸完全殺死他是困難的，非加熱食品本來就存在，即使在加熱食品中也存在由蠟狀芽孢桿菌嘔吐毒素造成的汙染的問題。蠟狀芽孢桿菌在世界範圍內以食物中毒菌聞名，即使在我國由於該菌引起的食物中毒也報告了多起。在1994年，從蠟狀芽孢桿菌分離、純化了該嘔吐毒素(命名為催吐素，セレウリド)，確定了其化學構造(Agat，N.，等FEMS Microbiol.Lett.121，31-34(1994))。隨之而來開發了利用HEp-2細胞檢測催吐素的方法(Agat，N.，等FEMS Microbiol.Lett.121，31-34(1994))。
弄清楚在食品以及其他檢體中有無嘔吐毒素(催吐素)在通過HACCP進行的食品製造管理上是非常重要的，在世界範圍內一直探求其檢測法的開發。然而，直到現在簡便而迅速地進行蠟狀芽孢桿菌的檢測以及催吐素檢測的方法也沒有開發出來。由於利用上述的HEp-2細胞的方法也需要熟練的技術，所以簡便而且正確的檢測，以及多個檢體同時處理是困難的。另外，檢體為患者的嘔吐物、糞便、食品或擦拭樣品時，連蠟狀芽孢桿菌的鑑定都必須進行經增菌培養、分離培養直至純培養、確認培養的操作。在各個培養階段需要的時間都各為18～24小時，總共需要的時間約4天。
本發明是在以上背景下完成的發明，目的在於提供在催吐素的檢測中可以利用的多肽、核酸等，以及利用他們的簡便、而迅速的催吐素的檢測法。

發明內容
本發明人鑑於上述目的，進行了不斷研究。結果首先發現參與催吐素生物合成的酶，並且在其鑑定中獲得了成功。當對編碼該酶的基因的鹼基序列和不產生催吐素的蠟狀芽孢桿菌保有的對應基因進行比較時，發現了在兩者之間存在不同序列，直至獲得了利用這一不同部分可以進行催吐素的檢測的想法。本發明是根據以上想法完成的發明，提供以下構成。
一種含有序列編號1的胺基酸序列的多肽，或含有序列編號1的胺基酸序列的一部分被改變後構成的序列、而且具有催吐素合成活性的多肽。
一種含有序列編號3的胺基酸序列的多肽，或含有序列編號3的胺基酸序列的一部分被改變後構成的序列、而且保持上述多肽所具有的有關催吐素合成的功能構造的多肽。
編碼[1]所述的任一種多肽的核酸。
編碼[2]所述的任一種多肽的核酸。
保有[3]或[4]所述核酸的載體。
用[5]所述載體轉化的轉化體。
含有序列6的鹼基序列中與催吐素合成活性直接有關的區域的至少部分的序列、或含有與上述區域的鹼基序列相互補的序列的至少部分的核酸。
含有序列7鹼基序列的至少部分、或含有與上述鹼基序列互補的序列的至少部分的核酸。
含有序列8的鹼基序列中與催吐素合成活性直接有關的區域的鹼基序列互補的序列的至少部分的核酸。
含有與序列編號9的鹼基序列互補的序列的至少部分的核酸。
被設計成可以對含有在編碼具有催吐素合成活性的多肽的DNA中直接與催吐素合成活性有關的區域的至少部分的DNA區域進行特異擴增的一組核酸。
一種將[7]～[10]任一項所述的核酸固定於不溶性支持體後構成的固相化核酸。
一種與催吐素合成酶特異結合的抗體。
一種抗體，其特徵是對含有序列編號1的胺基酸序列的多肽具有結合性、而對由序列編號2的胺基酸序列構成的多肽沒有結合性。
一種含有[7]～[10]任一項所述的核酸、[11]所述的一組核酸或[12]所述的固相化核酸的催吐素檢測用試劑盒。
一種含有[11]所述的一組核酸、DNA擴增用酶以及DNA合成試劑的催吐素檢測用試劑盒。
一種含有[13]或[14]所述抗體和抗原抗體反應用試劑的催吐素檢測用試劑盒。
一種包括研究檢體中的(a)或(b)存在的步驟的催吐素檢測方法(a)含有序列編號1的胺基酸序列的多肽，或含有序列編號1的胺基酸序列的一部分被改變後構成的序列、而且具有催吐素合成活性的多肽，(b)編碼(a)中任一種多肽的核酸。
一種包括以下步驟的催吐素檢測方法(i)以檢體中的DNA為模板，使用[11]所述的一組核酸進行DNA擴增反應的步驟，(ii)對被擴增的DNA進行檢測的步驟。
一種包括以下步驟的催吐素檢測方法(iii)以檢體中的DNA為模板製備cDNA的步驟，(iv)使用[11]所述的一組核酸進行DNA擴增反應的步驟，(v)對被擴增的DNA進行檢測的步驟。
一種包括以下步驟的催吐素檢測方法(I)使檢體與[13]或[14]所述抗體接觸的步驟，(II)I的步驟後，對抗原抗體反應物進行檢測的步驟。
[18]～[21]中任一項所述的催吐素檢測方法，其中作為前處理進行以下步驟(A)將檢體接種到蠟狀芽孢桿菌的增菌培養基中進行培養的步驟。
[18]～[21]中任一項所述的催吐素檢測方法，其中作為前處理進行以下步驟(A)將檢體接種到蠟狀芽孢桿菌的增菌培養基中進行培養的步驟，(B)對增殖的蠟狀芽孢桿菌進行溶菌或破碎的步驟。
另外，本發明中的DNA不限於雙鏈DNA，也可使用包括構成雙鏈的單鏈DNA(有義鏈和反義鏈)。另外在本發明的DNA中包含含有考慮了密碼子的簡併性的任意的鹼基序列的DNA。另外其形式也沒有限定，可以含有cDNA、基因組DNA、合成DNA。
另外，在本發明中所謂多肽指的是廣義上的多肽，即包括使用表現為多個胺基酸以肽鍵連接形成的肽在內的寡肽、狹義多肽以及蛋白質。
在本發明中，將蠟狀芽孢桿菌產生的嘔吐毒素稱為催吐素。
附圖的簡單說明

圖1是表示在本發明的實施例中對PCR擴增產物進行電泳後的凝膠染色後的狀態圖。從左邊的帶開始依次為對NC7401株、NC-T株、NC-G15株、NC327株、NC-1-55株(以上為催吐素產生株)、ATCC14579株、B-4ac株、PHLS2668株、PHLS4433株、NC1225株(以上為催吐素非產生株)、蘇雲金芽孢桿菌(HD73)、以及枯草芽孢桿菌(ATCC21332)的PCR反應後的溶液進行電泳後的結果。
圖2是表示對來自DNA濃度不同的樣品的PCR擴增產物進行電泳後的凝膠染色後的狀態圖。帶編號1、2、3和4分別為對來自300ng、30ng、3ng、以及0.3ng的樣品的PCR擴增產物進行電泳後的帶。
實施發明的最佳方式本發明的第1個方面涉及到具有催吐素合成活性的多肽(以下也稱為「催吐素合成酶」)。本發明中提供的催吐素合成酶含有序列編號1的胺基酸序列。就象下面實施例所示那樣，該酶是用蠟狀芽孢桿菌的cDNA文庫鑑定的。催吐素合成酶參與催吐素的合成，只出現在產生催吐素的蠟狀芽孢桿菌中。因此，檢體中該酶的存在可以反映有無催吐素產生。這樣一來，催吐素合成酶在作為催吐素檢測的指標方面是有用的。
另外，如果獲得與催吐素合成酶特異結合的抗體，利用該抗體提供免疫學方法也可以進行該酶的檢測、即催吐素的檢測。因此，在可以作為用於製備這樣的抗體的免疫源(抗原)利用方面，本發明提供的多肽(催吐素合成酶)也是有用的。
其中作為含有序列編號1的胺基酸序列的一部分被改變後構成的胺基酸序列的多肽(以下稱為「改變多肽」)只要是具有催吐素合成活性的，可以與上述多肽同樣在催吐素的檢測等中利用。作為這樣的多肽的例子，如關於含有序列編號1的胺基酸序列的多肽、保存與催吐素合成活性有關的立體構造的多肽。
這裡所謂的「胺基酸序列的一部分被改變」指的是在胺基酸序列中缺失、置換、附加、和/或插入1個或數個胺基酸。只要是保持催吐素合成活性，胺基酸序列的改變(變異)位置沒有特別限定，也可以在多個位置進行改變。改變的胺基酸數例如可以是相當於總胺基酸的10％以內的數，優選是相當於總胺基酸的5％以內的數。更優選的是相當於總胺基酸的1％以內的數。以上那樣改變的多肽可以用眾所周知的基因工程的手段進行製作。
象下面敘述的實施例所示的那樣，由序列編號1的胺基酸序列構成的多肽可以認為是由4個結構域構成的。在本發明中為了方便，從N末端一側開始依次將這些結構域稱為CRS1、CRS2、CRS3和CRS4。根據本發明人的研究結果，催吐素產生菌中特徵性的結構域是CRS3(1805位～2824位的胺基酸序列)和CRS4(2825位～3704位的胺基酸序列)。因此，可以認為這些結構域直接參與催吐素生物合成。由此可以認為為了製備對催吐素合成酶特異的抗體，含有上述特異結構域中的任一個或兩者的多肽可以成為特別有效的抗原。本發明也提供含有序列編號3、序列編號4、或序列編號5記載的胺基酸序列的多肽。另外在這些多肽中，只要是保持他們具有的與催吐素生物合成有關的功能構造，即使一部分胺基酸被改變也可以。
另外，只要是沒有特別限定，以下說明中，稱之為「催吐素生物合成直接相關的區域」指的都是序列編號1的胺基酸序列中1805位～3704位的區域，即CRS3和CRS4(對於DNA是編碼CRS3和CRS4的DNA區域)。
本發明的多肽中，若是存在於自然界中的，可以通過提取純化等操作作為天然的多肽製備。例如，可以從產生催吐素的蠟狀芽孢桿菌的菌體內製備。
另外，本發明的多肽(包括改變多肽)也可以用基因工程手法作為重組多肽製備。即，用編碼本發明的多肽的DNA轉化適當的宿主細胞，通過回收在轉化體內表達的多肽進行製備。回收的多肽可以根據目的進行適當純化。作為重組多肽製備時，可以進行各種修飾。例如，將編碼本發明的多肽與其他的適當DNA同時插入到載體中，可以得到本發明的多肽和上述另一個DNA編碼的肽或多肽連接的重組多肽。通過這樣的修飾，可以使重組多肽的提取、純化簡便，或附加生物學上的功能。
本發明的多肽也可以通過化學合成製備。例如，可以通過眾所周知的肽合成法-固相合成法進行合成。
本發明的第2個方面提供編碼上述本發明的多肽的核酸。作為這樣的核酸的具體例子，如具有序列編號6或序列編號7的鹼基序列的DNA、或序列編號8或序列編號9的RNA。或者是這些DNA中一部分被改變的DNA等。這裡的所謂「一部分被改變」指的是構成DNA或RNA的鹼基的一部分被缺失、置換、插入或附加。改變的鹼基數可以是例如1～100個、優選1～20個、更優選1～10個。
本發明的核酸可以用作對催吐素進行檢測時的樣品，即在給予有關有無催吐素存在的指標方面是有用的。另外，為了製備與上述本發明的多肽結合的抗體、即在催吐素的檢測中可以利用的抗體的抗原製備過程方面也是有用的。
以上的核酸可以適當利用可與編碼催吐素合成酶的基因(具有序列編號6的鹼基序列的DNA)特異雜交的探針、引物等從適當的基因組DNA文庫或cDNA文庫、或催吐素產生菌的菌體內提取液製備。另外也可以通過以至少編碼催吐素合成酶的基因的一部分作為模板，以dNTP(dATP、dGTP、dCTP、dTTP)作為原料的PCR法等進行合成。
為了製備本發明的DNA使用的基因組DNA文庫或cDNA文庫可以根據常規方法從蠟狀芽孢桿菌NC7401株製作。
本發明還提供保持上述本發明的DNA(包括改變DNA)的載體。只要是可以保持本發明的DNA的載體，可以使用任一種載體，但最好是根據目的(克隆、多肽的表達)，或考慮宿主細胞的種類之後選擇適當的載體。本發明的DNA向載體插入可以通過使用限制酶和DNA連接酶的眾所周知的方法(Molecular Cloning，ThirdEdition，1.84，Cold Spring Harbor Laboratory Press，New York)進行。
本發明還提供保持上述本發明的DNA(包括改變DNA)的轉化體。即涉及到用本發明的DNA轉化宿主細胞得到的轉化體。例如利用磷酸鈣法、電穿孔(Potter，H.等，Proc.Natl.Acad.Sci.U.S.A.81，7161-7165(1984))、脂轉染法(Felgner，P.L.等，Proc.Natl.Acad.Sci.U.S.A.84，7413-7417(1984))、微注射(Graessmann，M.Graessmann，A.，Proc.Natl.Acad.Sci.
U.S.A.73，366-370(1976))等眾所周知的基因導入方法將本發明的DNA導入到宿主細胞後進行轉化。用上述本發明的載體轉化宿主細胞後可以得到本發明的轉化體。根據目的可以使用各種各樣的宿主細胞，例如可以使用大腸桿菌等原核細胞、酵母等真核細胞。在利用大腸桿菌系時，作為表達載體可以使用pET-3c或pET-8c等pET載體(Novagen公司)、pBAD質粒(Invitrogen公司)、pGEX質粒(AmershamPharmacia biotech公司)等。
通過在適當的條件下對本發明的轉化體進行培養，可以大量生產本發明的DNA表達產物(多肽)，該表達產物可在催吐素合成酶的檢測用抗體的製備中利用。另外通過作為與由數個組氨酸構成的His-Tag、β-D-半乳糖苷酶、GST(穀胱甘肽S-轉移酶)、硫氧還蛋白、麥芽糖結合蛋白質、Myc、Xpress、FLAG等標記分子的融合蛋白質(肽)表達，可容易進行表達產物的純化。
本發明的第3個方面涉及到催吐素的檢測方法，其特徵是包括研究檢體中是否存在以下(a)或(b)的步驟。(a)含有序列編號1的胺基酸序列的多肽，或含有序列編號1的胺基酸序列的一部分被改變後構成的序列、而且具有催吐素合成活性的多肽，或(b)編碼(a)中任一種多肽的核酸。另外，由於檢體中催吐素的存在即意味著產生催吐素的蠟狀芽孢桿菌的存在，所以本發明中的「催吐素的檢測方法」可以作為與「產生催吐素的蠟狀芽孢桿菌的檢測方法」同義的概念使用。
研究(a)存在的方法沒有特別限定，可以利用使用對檢測對象的多肽特異的抗體的免疫學方法。
同樣，研究(b)存在的方法沒有特別限定，例如可以利用使用對編碼檢測對象多肽的核酸特異的核酸引物和/或核酸探針的方法，使用設計成對催吐素特異的核酸區域進行特異擴增的一組引物(核酸)的PCR法(聚合酶鏈式反應)以及其改變法或應用方法(PCR-RFLP限制片段長度多態性)法、RT-PCR(逆轉錄酶PCR)法等)，Soutern blot雜交法、點雜交法(Southern，E.，J.Mol.Biol.98，503-517(1975))，Northern blot法等。
以下給出了本發明提供的催吐素檢測方法的更具體的例子。首先，作為利用PCR法等的核酸擴增反應的例子，如包括(i)以檢體中的DNA為模板，使用一組核酸進行DNA擴增反應的步驟，該組核酸被設計成可以對含有編碼催吐素合成活性的多肽的DNA(例如由序列編號6的鹼基序列構成的DNA)中，與催吐素合成活性直接相關的至少一部分的DNA區域(例如由序列編號7的鹼基序列構成的DNA區域)進行特異擴增，以及(ii)對被擴增的DNA進行檢測的步驟的方法。另外作為利用RT-PCR法的方法，如包括(iii)以檢體中的mRNA為模板製備cDNA的步驟，(iv)使用一組核酸進行DNA擴增反應的步驟，該組核酸被設計成可以對含有編碼催吐素合成活性的多肽的DNA中與催吐素合成活性直接有關的至少一部分DNA區域進行特異擴增，以及(v)對被擴增的DNA進行檢測的步驟的方法。
由於必須是可通過PCR法等擴增得到的大小，所以由一組引物規定的擴增區域最好是約4,000bp以下。另外，當擴增區域過小時，由於對擴增產物和引物的二聚體難於進行區別，所以擴增區域最好是50bp以上。另外，為了有效進行擴增，擴增區域優選100bp～1,000bp大小。
作為在PCR法等中使雙鏈核酸熱變性時的溫度，例如可以是約90℃～約95℃，作為使引物雜交的退火溫度，例如可以為約37℃～約65℃，而作為聚合反應時的溫度例如可以為50℃～約80℃。在PCR法以及其變更方法等中可以將這些熱變性、退火、聚合作為一個循環反覆進行直至擴增到可以檢測到擴增產物為止。擴增產物的檢測可以利用瓊脂糖凝膠電泳進行。即，通過對酶反應液進行瓊脂糖凝膠電泳，可以確認擴增的核酸片段的存在及其長度。由該電泳結果可以判定在檢體中是否存在含有引物識別的序列的核酸，由此可以判定有無催吐素存在，即有無產生催吐素的蠟狀芽孢桿菌的存在。在擴增產物的檢測中不限定於瓊脂糖凝膠電泳，也可以利用其他電泳或各種層析。
作為在本發明的催吐素檢測方法中能夠利用的核酸(引物或探針用)，只要是在對編碼催吐素合成酶的基因進行特異檢測中可以利用的，沒有特別限定，例如至少含有序列編號6的鹼基序列中與催吐素合成活性直接有關的區域的一部分序列的核酸、或與上述區域的鹼基序列互補的序列的一部分的核酸。這裡所謂「與催吐素合成活性直接有關的區域」如上所述，具體指的是編碼CRS3和CRS4的區域，即具有序列編號7的鹼基序列的區域。在用檢體中的mRNA作為檢測對象時，在催吐素檢測方法中也同樣可以利用至少含有與序列編號8的鹼基序列中與催吐素合成活性直接有關區域的碳基序列互補的序列的一部分的核酸。更具體講，如至少含有與序列編號9的鹼基序列互補的序列的一部分的核酸。
至於探針、引物，可以根據解析方法使用合適的DNA片段或RNA片段。探針、引物的鹼基長度只要是能夠發揮各自功能的長度就可以，如果考慮到選擇性或檢測靈敏度和再現性，作為引物的鹼基長度為10bp以上、優選在15bp以上，具體來說如10～30bp左右，優選15～25bp程度。
另外，對於引物，只要是可以與擴增對象特異雜交、能夠擴增目的DNA片段，即使與作為模板的序列多少有些錯配也可以。錯配的程度可以是1～n個，優選1～5個，更優選1～3個。對於探針也同樣，在對檢測沒有影響的範圍內，即使對檢測對象的序列多少有點錯配也可以。
以下給出了在運用PCR法等伴隨特定DNA區域的擴增的方法的催吐素檢測方法中能夠利用的核酸(引物組)的具體例子。
引物組1有義鏈用引物5』-GGTGAATTGTGTCTGGGAGG-3』(序列編號10)反義鏈用引物5』-ATTTTTATTAAGAGGCAATG-3』(序列編號11)引物組2有義鏈用引物5』-GTCAAGATAAGAGGCTTCCGAATT-3』(序列編號12)反義鏈用引物5』-AATGGAATGACCACCAAGCT-3』(序列編號13)引物組3有義鏈用引物5』-AGGAAGTTCCGTTTGTGGAC-3』(序列編號14)
反義鏈用引物5』-CACATAACCTTTTGCAACTC-3』(序列編號15)引物組4有義鏈用引物5』-GGCGAACTATGTGTTGGTGG-3』(序列編號16)反義鏈用引物5』-TAAAGAGTCACCACCATAAG-3』(序列編號17)引物組5有義鏈用引物5』-ACGTCAGGCAGTACTGGAAA-3』(序列編號18)反義鏈用引物5』-TTCGATGCGGAATCCACGAA-3』(序列編號19)本發明中的核酸(引物、探針)可以通過磷酸二酯法等眾所周知的方法合成。另外，作為探針使用時的標記物質、標記方式可以採用眾所周知的物質。作為標記物質如32P等放射性同位素、異硫氰酸螢光素、異硫氰酸四甲基羅丹明等螢光物質，作為標記方法如使用T4DNA聚合酶或Klenow片段的3』末端標記法、切口移位法、隨機引物法(Molecular Cloning，Third Edition，Chapter 9，Cold SpringHarbor Laboratory Press，New York)等。
以下就利用免疫學方法的催吐素的檢測方法進行說明。作為利用免疫學方法的催吐素的檢測方法，如包括I)使檢體與對催吐素合成酶特異的抗體接觸的步驟，(II)I的步驟後，抗原抗體反應物的檢出步驟在內的方法。這裡的所謂「對催吐素合成酶特異的抗體」指的是具有與催吐素合成酶特異結合性的抗體，作為具體例子，如對含有序列編號1的胺基酸序列的多肽具有結合性、而對由序列編號2的胺基酸序列構成的多肽沒有結合性的抗體。可以使用的抗體的類別沒有特別限定，例如可以使用屬於IgG類、IgM類等的抗體。另外也可以使用Fab、Fab』、F(ab』)2，scFv、dsFv等抗體片段。
作為測定方法，如ELISA(酶聯免疫吸附定量法)法、放射免疫分析、FACS、免疫沉澱法、免疫印跡等定性的或定量的方法。另外作為抗原抗體反應的種類可以採用使檢體中的催吐素合成酶和另外添加的催吐素合成酶與催吐素合成酶特異的抗體競爭反應的方法(競爭法)，以及不使他們進行競爭反應的方法(非競爭法)。
作為對催吐素合成酶特異的抗體優選使用單克隆抗體。由於單克隆抗體的特異性高，所以可以進行高靈敏度測定。另外，利用使用對催吐素合成酶特異的而且識別互不相同的抗原表位的2種抗體的夾層法在靈敏度、特異性方面更好。
抗體可以進行固相化後使用。作為固相化使用的不溶性支持體，可以使用聚苯乙烯樹脂、聚碳酸酯樹脂、矽樹脂、尼龍樹脂等樹脂或玻璃等對水不溶性的物質，其材料沒有特別限定。抗體向這些不溶性支持體的負載可以通過物理吸附或化學吸附進行。
作為在免疫學測定法中可使用的標記物質，過氧化物酶、鹼性磷酸酶、β-D-半乳糖苷酶、葡萄糖氧化酶、葡萄糖-6-磷酸脫氫酶、以及微過氧化物酶等酶、異硫氰酸螢光素(FITC)、異硫氰酸四甲基羅丹明(TRITC)、以及銪等螢光物質、氨基苯二醯肼、異氨基苯二醯肼、以及吖啶鎓衍生物等化學發光物質、NAD等輔酶、生物素以及131I和125I等放射性物質等可以作為標記物質使用。特別是作為標記物質使用生物素，使用螢光色素或酶標記的抗生物素蛋白(例如抗生物素蛋白過氧化物酶)反應的方法，可以進行更高靈敏度的測定。
與催吐素合成酶特異結合的單克隆抗體可以通過常規方法獲得。以下給出了一個單克隆抗體的製作方法的例子。首先取得催吐素合成酶，作為抗原免疫小鼠等動物。然後從被免疫的動物摘出抗體產生細胞，使該細胞與骨髓瘤細胞融合得到雜交瘤細胞。接下來，使該雜交瘤單克隆化後，選擇產生與催吐素合成酶特異結合的克隆。
作為抗原可以使用從產生催吐素的蠟狀芽孢桿菌的菌體內分離、純化的催吐素合成酶。另外也可以使用利用用編碼催吐素合成酶的鹼基序列，通過來自大腸桿菌的表達體系得到的重組多肽。
作為免疫方法，例如可以採用將上述抗原與弗氏完全或不完全佐劑混合，進行乳化，注射到小鼠等的腹腔內、皮下或肌肉，隔一定時間注射一次，進行數次注射的方法。作為免疫的動物可以使用小鼠以及大鼠、倉鼠、兔、豚鼠、雞、綿羊、山羊等。免疫完成後，取出免疫的動物的脾臟，取得抗體產生細胞。也可以從淋巴節、末梢血液等採取抗體產生細胞。
使用的骨髓瘤細胞的種類沒有特別限定，選擇與免疫使用的動物的相關的合適的種類。最好是使用來自與抗體產生細胞同種動物的骨髓瘤細胞，例如當使用小鼠時可以使用骨髓瘤細胞株PAI。細胞融合，例如可以通過按照一定比例將抗體產生細胞與骨髓瘤細胞混合，然後加入聚乙二醇後攪拌處理進行細胞融合。另外也可以使用用電脈衝進行細胞融合。
在只選擇進行了細胞融合的雜交瘤時，可以運用使用一般的HAT培養基(按給定比例含有次黃嘌呤、氨基蝶呤、胸腺嘧啶的選擇培養基)的方法。含有雜交瘤的培養液為了以後的選擇，可以在96孔板中培育。
然後取各個容器內的培養上清，通過使用催吐素合成酶的ELISA法等選擇產生抗催吐素合成酶的抗體的雜交瘤。抗體陽性的容器內的雜交瘤通過有限稀釋法進行克隆，可以得到單克隆化的雜交瘤細胞株。
通過對雜交瘤的培養液進行純化可以得到所期望的抗體。另外使雜交瘤增殖到所期望的數以上後，將他們移植到動物(例如小鼠)的腹腔內，使其在腹水內增殖，通過純化腹水，也可以獲得所期望的抗體。在上述培養液的純化或腹水的純化中適合採用使用蛋白G、蛋白A等的親和層析。另外，也可以使用將抗原固相化的親和層析。另外也可以使用離子交換層析、凝膠過濾層析、硫銨分級、以及離心分離等方法。這些方法可以單獨或任意組合使用。
通過以上方法得到的抗體是否特異識別催吐素合成酶，例如可以通過使用將催吐素合成酶固相化的板的ELISA法進行確認。
在本發明的催吐素檢測方法中可以將利用的核酸(引物或探針)固定於不溶性支持體後使用。同樣，在本發明的催吐素檢測方法中也可以將可利用的抗體固定於不溶性支持體後使用。可以將固定化中使用的不溶性支持體事先加工成片狀、顆粒狀等，使用這些固定化核酸或固定化抗體可以簡便地進行檢體中的催吐素的檢測。
本發明的另一個方面提供在催吐素檢測中可以利用的試劑盒。即，使用在催吐素的檢測中可以利用的核酸(含有一組的核酸、固相化核酸)構建催吐素檢測用試劑盒。在催吐素檢測用試劑盒中除了這些核酸之外，還可以含有核酸擴增用酶(例如PCR法中使用的DNA合成酶)以及作為底物的核酸(dATP、dCTP、dGTP、dTTP)、反應用的試劑等。另外，作為標準物質可以含有催吐素合成酶(可以是部分純化品)、催吐素產生菌株的菌體內提取物(可以是進行了部分純化提取物)。
利用以上的催吐素檢測用試劑盒，以檢體中的核酸作為靶可以檢測催吐素，也可以構建以檢體中的多肽、即催吐素合成酶作為靶，用於對催吐素進行檢測的試劑盒。在這樣的試劑盒中含有對上述的催吐素合成酶特異的抗體(包括固相化抗體)。此外，也可以構建包含與該抗體結合的第二抗體、抗原抗體反應用試劑(緩衝液、發色底物、發色試劑、發色反應終止液等)等的試劑盒。另外作為標準物質可以含有催吐素合成酶(可以是部分純化品)、催吐素產生菌株的菌體內提取物(可以是進行了部分純化的提取物)。
提供給本發明的催吐素檢測方法的檢體沒有特別限定，作為檢體例如可以使用各種食品、人或動物的嘔吐物或糞便、或擦拭樣品等。這些檢體可以預先進行用溶菌酶等酶處理、加壓處理、加熱處理或超聲處理等。通過這些處理，檢體中的菌體被溶菌或破碎。但是當預料在採取檢體時，檢體中的蠟狀芽孢桿菌的細胞膜破碎時，就不必進行溶菌處理等。
對於從檢查對象採取的樣品最好是用蠟狀芽孢桿菌的增殖培養基(選擇培養基)預先進行培養。通過採用該培養步驟，可以進行可靠性更高的檢測。
另外，檢查對象為液狀時，可以將採取的樣品用於直接溶菌等處理或培養步驟，當檢查對象為固體狀時，優選是用適當的溶劑對菌體進行提取後，用於這些處理。
以下用實施例對本發明進行更詳細說明。
實施例1從蠟狀芽孢桿菌基因組DNA文庫克隆嘔吐毒素(催吐素)合成酶基因使用來自蠟狀芽孢桿菌NC7401株(保管在名古屋市衛生研究所、國立醫藥品食品衛生研究所)的EMBL3(Promega公司生產)製作噬菌體文庫。對得到的約400個白色蝕斑進行篩選，作為在不藉助於核糖體的胺基酸合成酶中特異存在的區域，用BSCI(GGAATTCCTTAAAIGCIGGAGGAGCITATGTGCCGCTT GATCC序列編號20)以及II(GGAATTCCTTTIGGITTICCIGTTGTICCI GAIGTGTAAAT序列編號21)作為引物(Kathrin，M.，等，FEMSMicrobiol.Lett.135，295-303(1996))特異擴增的DNA片段通過Southern雜交法進行分析。另外對通過作為模板使用NC7401株的染色體DNA，作為引物使用BSC I和II的PCR法擴增的DNA片段用標記物標記試劑盒(Roch Diagnostics公司生產)進行標記的探針進行分析。分析結果，選擇多個表達量大的插入DNA，分別用限制性內切酶SaIl切出，亞克隆到克隆載體pHSG299(寶酒造株式會社生產)的多克隆位點。
實施例2嘔吐毒素(催吐素)合成酶cDNA的序列分析亞克隆的各個DNA片段的序列通過使用自動測序儀(AppliedBiosystems公司生產)循環測序反應進行分析。考慮到重複的序列後，通過對各個DNA片段的序列信息進行分析，決定催吐素合成酶的全長DNA序列以及胺基酸序列(序列編號1)。當對該序列進行詳細研究時，發現催吐素合成酶由合成各個胺基酸的的4個結構域構成，N末端一側的2個結構域雖然也廣泛地存在於不產生催吐素的蠟狀芽孢桿菌株中，但C末端一側的2個結構域是產生催吐素的菌株所特異的。
實施例3利用PCR法進行催吐素的檢測(3-1)檢體的製備從圖1的表表示的芽孢桿菌(催吐素產生株和非產生株各5株)按照以下順序製備檢體。作為對照組使用蘇雲金芽孢桿菌(Bacillusthuringienesis)、以及枯草芽孢桿菌(Bacillus subtilis)。
將各個菌體分別接種到適當的增菌培養基(LB培養基)，於37℃下在好氧條件下培養過夜，通過離心操作從培養後的1.5ml培養基中回收菌體。將回收的菌體用10mMTris-HCl緩衝液(pH7.5)清洗一次後，懸浮於0.5ml溶解了終濃度為1mg/ml的溶菌酶的同一緩衝液中。在該狀態下於37℃下放置10分鐘，使其溶菌。然後，向溶菌液中添加等量被上述緩衝液飽和的酚，進行充分攪拌。離心處理後，回收上層液，進行醇沉澱處理，使核酸成分沉澱。將得到的沉澱物溶解於1ml上述緩衝液，作為以下檢測方法中的檢體。
(3-2)PCR用引物的合成以序列編號6給出的催吐素合成酶的鹼基序列信息為基礎，選擇對催吐素產生株特異的序列，化學合成以下所示引物(寡核苷酸)。
有義鏈用引物5』-GGTGAATTGTGTCTGGGAGG-3』(序列編號10)反義鏈用引物5』-ATTTTTATTAAGAGGCAATG-3』(序列編號11)(3-3)PCR法向上述各個檢體3μl加入滅菌蒸餾水16.05μl、10×反應用緩衝液3μl、dNTP溶液4.8μl、有義鏈用引物1.5μl、反義鏈用引物1.5μl、以及耐熱性DNA聚合酶0.150μl，將總量調整到約30μl的反應液。而10×反應用緩衝液的組成為500mM KCl、100mM Tris-HCl(pH8.3)、15mM MgCl2、0.1％(w/v)明膠，dNTP溶液是使終濃度各為1.25mM的dATP、dCTP、dGTP和dTTP混合的溶液。另外各個引物是(3-2)中得到的化學合成純化品的水溶液(50DU/ml)。至於耐熱性DNA聚合酶使用TaqDNA聚合酶(5unit/mlPerkin Elmer Cetus公司生產)。
PCR反應條件如下。即熱變性94℃1分鐘，退火55℃1分鐘，聚合反應72℃1分鐘。由熱變性開始經退火直至聚合反應的過程作為1個循環，共進行35次循環。另外，使用DNA Thermal Cycler(Perkin Elmer Cetus公司生產)進行PCR反應。
(3-4)PCR擴增產物的檢測為了檢測由PCR反應液擴增的DNA片段，在以下條件下進行瓊脂糖電泳。作為瓊脂糖凝膠使用凝膠濃度為2％(w/v)的凝膠。使用溴乙錠(0.5μg/ml)進行電泳後的凝膠染色。電泳條件為加100V電壓，電泳30分鐘。其他的電泳條件以及操作方法根據MolecularCloning，Third Edition，Cold Spring Harbor LaboratoryPress，New York記載的方法。
圖1表示染色後的凝膠。在圖1中，從左邊的帶開始依次為對NC7401株、NC-T株、NC-G15株、NC327株、NC-1-55株(以上為產生催吐素株)、ATCC14579株、B-4ac株、PHLS2668株、PHLS4433株、NC1225株(以上為不產生催吐素的菌株)、蘇雲金芽孢桿菌(HD73)、以及枯草芽孢桿菌(ATCC21332)的PCR反應後的溶液進行電泳後的結果。就象圖1所表示的那樣，可知在催吐素產生菌株(帶1～5)可以得到約450bp的PCR擴增產物。而在催吐素非產生菌株和蘇雲金芽孢桿菌HD73、以及枯草芽孢桿菌ATCC21332中沒有檢測到相當於該PCR擴增產物的帶。由以上結果可以確認通過本實施例的方法可特異進行催吐素產生菌株的檢測、即催吐素的檢測。
實施例4利用PCR法進行催吐素產生菌的微量檢測(4-1)檢體的DNA量的計算使用圖1表示的蠟狀芽孢桿菌NC7401株，用實施例的(3-1)給出的方法製備檢體，得到純化DNA標準品。然後通過測定波長260nm的吸光度計算出該標準品中的DNA量。
(4-2)PCR擴增產物的檢測以(4-1)求出的DNA量作為參考，對檢體進行稀釋，製備分別含有300ng(分子數約1×102)、30ng(分子數約1×101)、3ng(分子數約1)、0.3ng(分子數約1×10-1)的樣品。利用這些樣品通過實施例3的(3-2)和(3-3)給出的方法進行PCR，然後用(3-4)給出的方法進行PCR擴增產物的檢測。圖2給出了對PCR擴增產物電泳後的凝膠進行染色後的狀態。道編號1、2、3和4數分別對應來自300ng、3ng、3ng以及0.3的樣品的PCR擴增產物進行電泳後的道。道3，即使在使用含有3ng的DNA的樣品時也可以確認目的帶。這意味著可以對相當於蠟狀芽孢桿菌染色體的1～數個分子的DNA量進行檢測，由此可認為理論上如果在檢體中存在大約幾個催吐素產生蠟狀芽孢桿菌，也可以其存在、即催吐素進行檢測。
本發明並不受上述發明的實施方式以及實施例的說明的任何限定。只要是沒有脫離開專利申請範圍的記載，本領域技術人員容易想到的範圍中種種變化方式都包括在本發明中。
產業上利用的可能性本發明提供蠟狀芽孢桿菌產生的嘔吐毒素(催吐素)的胺基酸序列以及鹼基序列。利用這些序列，使用核酸探針或抗體有可能進行催吐素的檢測。通過使用對催吐素特異的抗體或核酸探針，對催吐素可以簡便而且迅速地進行檢測。在實施例中給出的本發明的檢測方法的一個例子中，對檢體中的核酸進行擴增的反應所需要的時間約3小時，直至獲得後來的檢測結果大約為30分鐘，可以在極短的時間內對到目前為止不能檢測的催吐素進行檢測。
另外利用本發明的方法可以對催吐素進行高靈敏度檢測。這意味著可以對少量檢體進行檢測，以及可以使檢體的前處理簡便化。另外通過本發明的催吐素檢測方法不僅可以對催吐素直接進行檢測，而且可以對催吐素合成酶的有無進行研究，由研究結果判別催吐素是否存在於檢體中。作為在蠟狀芽孢桿菌中產生催吐素的菌株的共通的特徵，認為是具有在本發明中鑑定的催吐素合成酶活性，即催吐素產生株必然有催吐素合成酶基因。然而，由於不知道在其他生物種中是否產生與催吐素同源的毒素，所以通過將催吐素合成酶或編碼該酶的基因作為檢測對象，可以有選擇地或特異地對檢體中的催吐素的存在(催吐素產生菌的存在)進行檢測。因此，可以得到可靠性高的結果，適用於食品檢查、臨床檢查。
序列表110股份有限公司生物控制研究所OHTA，MichioAGATA，Norio120催吐素合成酶，其基因，和催吐素的檢測方法130P0202401150JP P2002-1423981512002-05-1716021170PatentIn version 3.121012113704212PRT213蠟狀芽孢桿菌4001Phe Ile Lys Ser Met Asn Gln Leu Gly Lys Ser Lys Asn Leu His Asn1 5 10 15Gly Gly Met Met Glu Met Lys Arg Val Glu Glu His Asp His Ile His20 25 30Val Leu Asn Glu Ile Glu Asn Glu Cys Glu Arg Arg Tyr Gly Arg Ser35 40 45Asn Ile Ala Ile Met Leu Glu Lys His Gly Val His Glu Gln Pro Leu50 55 60His Ile Glu Asp Leu Phe His Glu Val Glu Met Gln Glu His Ser Arg65 70 75 80
Val Ser Arg His Glu Thr Val Leu Met Thr Asp Lys Gln Cys Ile Asp85 90 95Glu Ser Gly Lys Pro Leu Ala Leu Arg Phe Gly Glu Pro Leu His Leu100 105 110Asp Asp Cys Thr Pro Lys Thr Leu Gln Glu Ile Leu Lys Arg Ala Ala115 120 125Lys Gln Ala Lys Asp Lys Gly Met Thr Phe Val Tyr Glu Asp Gly His130 135 140Glu Glu Tyr Leu Ser Tyr Gln Glu Met Leu Ala Asp Ala Glu Arg Leu145 150 155 160Leu Lys Gly Leu Arg Asn Leu Gly Ile Gln Pro Gly Glu Ser Ile Leu165 170 175Phe Gln Phe Lys Asp Asn Lys His Phe Val Thr Ala Phe Trp Ala Cys180 185 190Ile Leu Gly Gly Phe Leu Pro Thr Pro Leu Gly Thr Ala Pro Ile Tyr195 200 205Ser Glu Gln Asn Ala Gln Val Leu Lys Leu Tyr Asn Thr Trp Gln Leu210 215 220Leu Glu Gln Pro Ile Ile Leu Thr Glu Phe Glu Leu Lys Glu Glu Ile225 230 235 240Ala Ala Ile Arg Thr Thr Leu Gln Arg Gln Glu Ile Val Ile His Ser245 250 255
Ile Glu Asn Val Met Asp Thr Ala Arg Asp Thr Asn Trp Phe Pro Cys260 265 270Thr Glu Asp Thr Ile Val Leu Asn Leu Leu Thr Ser Gly Ser Thr Gly275 280 285Val Pro Lys Cys Val Gln His Lys Ser Lys Ser Ile Ile Ala Arg Thr290 295 300Val Ser Asn Cys Ile Asp Arg Gln Leu Asp Glu Lys Glu Val Ser Leu305 310 315 320Asn Trp Met Pro Leu Asp His Val Gly Gly Ile Val Met Cys His Ile325 330 335Arg Asp Thr Tyr Leu Met Cys Gln Gln Val Asn Cys Leu Ile Ser Ala340 345 350Phe Ile Glu Asn Pro Leu Asn Trp Leu His Trp Ile Asp Ala Tyr Ser355 360 365Ala Thr Phe Thr Trp Ala Pro Asn Phe Ala Phe Ser Leu Ile Asn Gln370 375 380Tyr Glu Glu Glu Ile Lys Ser Ser Ser Trp Asn Leu Ser Ser Met Arg385 390 395 400Tyr Ile Val Asn Gly Gly Glu Ala Val Ile Ser Ser Val Gly Met Lys405 410 415Phe Leu Gln Leu Leu Gln Gln His Gln Leu Pro Ser Asn Cys Leu Ile420 425 430
Pro Thr Phe Gly Met Ser Glu Val Ser Ser Gly Ile Ile Glu Cys His435 440 445Ser Phe Tyr Thr Gln Thr Thr Asn Thr Gly Met Leu Tyr Val Asp Lys450 455 460Asn Ser Leu Asp Gly Asn Leu Gln Phe Thr Tyr Glu Gly His Gln Asn465 470 475 480Ala Ile Val Phe Thr Glu Val Gly Arg Pro Met Pro Gly Ile Gly Ile485 490 495Arg Ile Val Asp Glu Asp Asn Gln Cys Leu Ser Glu Asp Arg Ile Gly500 505 510Arg Phe Gln Ile His Gly Pro Thr Val Met Asn Gly Tyr Phe Lys Asn515 520 525Asp Glu Ala Asn Ala Glu Ser Phe Thr Glu Asp Gly Trp Phe Asp Ser530 535 540Gly Asp Leu Gly Phe Ile His Asn Gly Asn Leu Val Ile Thr Gly Arg545 550 555 560Lys Lys Asp Met Ile Val Val His Gly Ala Asn Tyr Tyr Asn Tyr Glu565 570 575Ile Glu Ala Leu Val Glu Gln Val Pro Gly Val Glu Thr Thr Phe Val580 585 590Cys Ala Thr Ser Val Lys Ser Ala Glu Gly Ala Glu Glu Leu Ala Ile595 600 605
Phe Phe Val Pro Val Ile Asn His Val Ser Val Met Phe Ala Thr Met610 615 620Gln Gln Ile Lys Gln Ile Val Ala Arg Lys Met Gly Ile Thr Pro Lys625 630 635 640Val Ile Ile Pro Ile Gln Lys Glu Ala Phe Phe Lys Thr Asp Ser Gly645 650 655Lys Ile Thr Arg Asn Ala Phe Gln Lys Gln Phe Glu Asn Gly Ala Tyr660 665 670Arg Glu Ile Thr Gln Lys Ile Asp Cys His Leu Gln Asn Glu Lys Thr675 680 685Leu Ser Gln Trp Phe Tyr Arg Glu Lys Leu Val Glu Ser Lys Leu Gly690 695 700Lys Ser Val Ser Ser Gln Lys Glu Thr Tyr Val Phe Phe Arg Gln Gly705 710 715 720Lys Ser Phe His His Val Leu Lys Glu Lys Leu Thr Gln His Ser Val725 730 735Val Ile Val Asp Val Gly Glu Thr Phe Gly Glu Ile His Pro Asn His740 745 750Tyr Gln Ile Asn Pro Lys Asn Lys Met Asp Tyr Val Arg Leu Phe Glu755 760 765Glu Leu Ala Lys Arg Asn Val Glu Asp Gln Val Phe His Leu Leu His770 775 780
Ala Trp Asn Tyr Cys Asp Thr Val Pro Thr Phe Arg Ser Val Glu Asp785 790 795 800Leu Ala Asn Ala Gln Tyr Leu Gly Val Phe Ser Val Met Phe Ala Leu805 810 815Gln Ala Ile Met His Ala Lys Leu Pro Leu Arg Arg Val Thr Val Ile820 825 830Ala Thr Asn Ser Val Gly Leu Glu Ala Lys Glu Met Asn Tyr Ser Cys835 840 845Ser Thr Leu G1u G1y Tyr Val Lys Thr Leu Pro Ala Glu Phe Glu Asn850 855 860Leu Gln Val Lys Tyr Ile Asp I1e G1u G1y Lys Asp I1e G1n Phe Asp865 870 875 880Thr Glu Thr Val Trp Lys Glu Leu Gln G1n Gln G1u Thr I1e Pro Val885 890 895Val Leu Tyr Arg Asp Glu Lys Arg Tyr Lys Ile Gly Leu G1u Lys Val900 905 910Pro Met Leu Glu Gln Lys Glu Lys Asn Ile Pro Phe Gln Gln Gln Gly915 920 925Phe Tyr Ile Ile Thr Gly Gly Leu Gly Gly Leu Gly Thr Leu Val Ala930 935 940Lys Leu Leu Leu Glu Arg Tyr Ser Ala Asn Val Leu Leu Leu Gly Arg945 950 955 960
Thr Glu Ile Glu Thr Asn Ala Glu Lys Met Arg Leu Leu Asp Ser Leu965 970 975Lys Glu Tyr Glu Gln Tyr Gly Gly Thr Val Gln Tyr Lys Met Cys Asn980 985 990Val Met Asp Leu Asp Ala Met Arg Lys Val Val His Ser Gln Glu Glu995 10001005Arg Leu Gln Gln Lys Val Asn Gly Ile Ile His Leu Ala Gly Ile101010151020Ile Gln Glu Ile Leu Ile Glu Lys Gln Thr Glu Lys Glu Leu His102510301035Ala Met Phe Glu Ala Lys Val Tyr Ala Ser Trp Val Leu His Glu104010451050Ile Val Lys Glu Arg Gln Asp Cys Leu Tyr Ile Thr Thr Ser Ser105510601065Ala Arg Thr Leu Leu Pro Gly Met Thr Ile Ser Ala Tyr Cys Ser107010751080Ala Asn Arg Phe Val Glu Asn Phe Ala Tyr Tyr Gln Arg Ser Gln108510901095Asn Val Asn Ser Tyr Cys Phe Ser Trp Ser Phe Trp Asn Glu Ile110011051110Gly Met Gly Thr Asn Leu Leu Ile Lys Asn Ala Leu Ile Ala Lys111511201125
Gly Phe Gln Leu Ile Asp Asp Gln Lys Gly Ile Tyr Ser Leu Leu113011351140Ala Gly Leu Lys Gly Asn Glu Pro Asn Val Phe Val Gly Ile Asn114511501155His Glu Lys Glu Glu Met Ala His Leu Ile Gly Thr Glu Glu Gln116011651170Glu Thr Gln Gln Leu Thr Ile Tyr Ile Thr Pro Glu Tyr Leu His117511801185Ile Leu Glu Glu Val Phe Ser Ile Leu Asn Arg Glu Glu Phe Gly119011951200Gly Leu Glu Lys Glu Ile Val Ile Leu Pro Lys Leu Pro Leu Asp120512101215Glu Tyr Gly Lys Val Asp Gln Thr Arg Leu Ala His Ala Ser Asp122012251230Ser Arg Phe Gly Lys Lys Gln His Ile Val Pro Arg Asn Asp Ile123512401245Glu Glu Lys Ile Ala Phe Ile Trp Glu Gly Leu Leu Asn Lys Lys125012551260Asp Ile Ser Val Leu Asp His Phe Phe Glu Leu Gly Gly Asp Ser126512701275Leu Lys Ala Thr Gln Met Ile Ser Ala Leu Lys Lys Asn Phe Ala128012851290
Val Thr Ile Thr Gln Gln Glu Phe Phe Gln Ser Ser Thr Val Glu129513001305Glu Leu Ala Ser Leu Val Glu Lys Lys Leu Ser Arg Thr Arg Thr131013151320His Glu Met Asp Ile Val Thr Phe Ser Asp Arg Gly Asn Val Val132513301335Glu Met Ser Ser Ala Gln Lys Arg Gln Trp Phe Leu Tyr Glu Met134013451350Asp Arg Glu Asn Pro Tyr Tyr Asn Asn Thr Leu Val Ile Arg Leu135513601365Thr Gly Glu Ile His Leu Pro Ile Leu Arg Ser Ser Ile Ile Glu137013751380Leu Val Asn Lys His Glu Thr Leu Arg Thr Thr Phe Val Met Val138513901395Asp Gly Ile Pro Ser Gln Ile Ile Ala Asp Glu Glu Leu Val Glu140014051410Ile Glu Glu Ile Asp Leu Lys His Leu Ser Ala Glu Glu Thr Leu141514201425Gln Lys Leu Glu Gly Leu Arg Gln Arg Glu Ala Asn Thr Ala Phe143014351440Lys Ile Glu Asn Ser Ala Phe Arg Ala Lys Val Ile Leu Ile Asp144514501455
Glu Lys Arg Val Glu Ile Leu Leu Ser Val His His Ile Val Ser146014651470Asp Gly Trp Ser Met Gly Ile Leu Val Lys Asp Ile Ala Glu Ile147514801485Tyr Glu Asp Ile Arg Gln Trp Gly Glu Ser Lys Gln Glu Pro Leu149014951500Pro Ile Gln Tyr Ala Asp Tyr Thr Leu Trp Gln Asn Glu Phe Met150515101515Lys Gly Glu Glu Phe Ser Lys Gln Leu Ser Tyr Trp Lys Glu Lys152015251530Leu Ala Glu Asp Ile Pro Val Leu Asp Leu Pro Leu Asp Lys Pro153515401545Arg Pro Pro Ile Gln Thr Tyr Arg Gly Lys Val Lys Thr Phe Thr155015551560Leu His Glu Asn Met Thr Arg Met Leu Lys Glu Ile Cys Gln Glu156515701575Glu Glu Cys Thr Leu Phe Met Leu Leu Leu Ser Ala Phe Ser Ser158015851590Leu Leu His Arg Tyr Thr Gly Gln Glu Asp Leu Val Val Gly Ser159516001605Leu Val Ala Asn Arg Asn Arg Glu Gln Ile Glu Lys Leu Ile Gly161016151620
Phe Phe Val Asn Thr Leu Pro Leu Arg Ile Asn Leu His Arg Glu162516301635Met Gln Phe Thr Glu Leu Leu Ser Gln Val Lys Lys Thr Thr Ile164016451650Asp Ala Tyr Asp His Gln Asp Val Pro Phe Glu Leu Leu Val Asp165516601665Glu Leu Gln Ile Glu Arg Asp Ser Ser Arg Asn Ala Leu Phe Gln167016751680Val Leu Phe Val Leu Gln Asn Ala Gln Leu Gln Ala Val Asp Leu168516901695Glu Lys Ala Thr Met Glu Leu Glu Ile Leu Asp Ser Asp Thr Ala170017051710Lys Phe Asp Met Ser Val Gln Ile Phe Glu Leu Glu Asp Thr Leu171517201725Ser Ile Lys Leu Glu Tyr Asn Thr Asp Leu Phe Phe Asp Asp Thr173017351740Ile Glu Arg Phe Leu Ala His Tyr Glu Thr Ile Leu Ala Ser Val174517501755Ile His Asn Gln Lys Ala Lys Ile Gly Glu Leu Ser Ile Leu Pro176017651770Gln Ser Glu Tyr Thr Lys Leu Val Ser Glu Trp Asn Glu Lys Ser177517801785
Ala Thr Tyr Asn Gly Asn Gln Cys Ile His Glu Leu Phe Glu Ala179017951800Ala Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn180518101815Lys Glu Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu182018251830Ala His Lys Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val183518401845Gly Val Leu Cys Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu185018551860Ala Val Leu Lys Ala Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala186518701875Tyr Pro Met Gln Arg Thr Glu Tyr Val Leu Gln Asn Ser Glu Ala188018851890Thr Ile Leu Leu Thr Lys Glu Cys Tyr Leu Lys Glu Ser Leu Asp189519001905Phe Glu Gly Glu Val Phe Tyr Leu Asp Asp Ala Arg Leu Phe Glu191019151920Gly Asp Arg Arg Asp Leu Gln Asn Ile Asn Asn Pro Thr Asn Leu192519301935Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr Gly Asn Pro Lys Gly194019451950
Val Met Val Ala His Gln Ser Val Val Asn Leu Leu Leu Asp Leu195519601965Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His Leu Leu Lys197019751980Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe Gly Trp198519901995Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp Glu200020052010Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val201520202025Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln203020352040Ala Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr204520502055Ile Ile Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg206020652070Leu Tyr Ala His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly207520802085Pro Thr Glu Gly Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys209020952100Glu Met Asn Val Ala Asn Val Pro Ile Gly Lys Pro Leu Ser His210521102115
Val Glu Thr Tyr Ile Leu Asp Gln Asn Asn Gln Ile Val Pro Ile212021252130Gly Val Pro Gly Glu Leu Cys Leu Gly Gly Ile Cys Val Ala Lys213521402145Gly Tyr Met Lys Glu Pro Val Leu Thr Glu Glu Lys Phe Val Val215021552160Asn Pro Met Lys Gln Ser Glu Arg Met Tyr Arg Thr Gly Asp Leu216521702175Val Arg Trp Leu Ala Asp Gly Asn Ile Glu Tyr Leu Gly Arg Ile218021852190Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Gly Glu219522002205Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val Gln Thr Ile221022152220Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile Val Ala222522302235Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu His224022452250Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val225522602265Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg227022752280
Lys Gln Leu His Ser Val Asp Leu Tyr Glu Thr Ser Met Asp Thr228522902295Val Ile Val Gly Pro Arg Asn Glu Lys Glu Ala Met Leu Ser Val230023052310Ile Trp Gln Glu Leu Leu Gly Leu Glu Asn Ile Ser Val His Asp231523202325Asn Phe Phe Lys Leu Gly Gly His Ser Ile Asn Ala Thr Gln Leu233023352340Val Ser Lys Ile Tyr Ser Val Cys Arg Val Arg Met Pro Leu Lys234523502355Asn Val Phe Gln Tyr Thr Thr Leu Ala Thr Met Ala Arg Val Leu236023652370Glu Glu Leu Leu Val Ser Ala Val Asp Glu Val Ala Val Thr Thr237523802385Glu Arg Ile Pro Lys Ile Leu Pro Arg Thr Tyr Tyr Asp Leu Ser239023952400Tyr Ser Gln Gln Arg Ile Tyr Phe Leu Ser Thr Met Glu Lys Glu240524102415Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp Asp Ile Tyr Gly Lys242024252430Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln Leu Leu Met Lys243524402445
Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val Asp Gly Lys245024552460Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val Gln Phe246524702475Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu Val248024852490Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala249525002505Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly251025152520Glu His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly252525302535Trp Ser Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp254025452550Ala Phe Val Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile255525602565Ser Ile Met Asp Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu257025752580Asp Gln Asp Asp Arg Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu258525902595Leu Pro Thr Leu Glu Leu Pro Thr Asp Arg Gln Arg Pro Leu Leu260026052610
Lys Thr Tyr His Gly Asp Thr Leu Ser Tyr Lys Val Asn Ser Gln261526202625Leu His Gln Lys Leu Lys Asp Phe Ser His Ala Asn Gly Val Thr263026352640Met Phe Met Thr Leu Leu Thr Ala Tyr Asn Ile Met Leu Asn Lys264526502655Leu Thr Asn Glu Thr Asp Ile Val Val Gly Ser Pro Val Ala Gly266026652670Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly Met Phe Val Asn267526802685Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro Thr Val Asp269026952700Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala Tyr Asn270527102715His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp Pro272027252730His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly273527402745Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val275027552760Arg Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr276527702775
Leu His Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu278027852790Tyr Asn Thr Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln279528002805Asn His Phe Glu Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr281028152820Lys Arg Ile Ser Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr282528302835Glu Ile Leu Glu Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His284028452850Glu Ser Val Ala Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln285528602865Ala Lys Leu Ala Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala287028752880Glu Leu Ser Glu Lys Ala Asn Ala Leu Ala His Thr Leu Lys Arg288528902895Arg Gly Val Ala Gln His Asp Val Val Gly Ile Val Ala Glu Arg290029052910Ser Pro Glu Thr Ile Ile Gly Ile Leu Ala Ile Leu Lys Val Gly291529202925Ala Ile Tyr Leu Pro Ile Asp Pro Lys Leu Pro Gln Leu Thr Leu293029352940
Gln His Ile Trp Arg Asp Ser Gly Ala Lys Val Leu Leu Gly Lys294529502955Asn Glu Thr Thr Val Glu Val Gly Lys Glu Val Pro Phe Val Asp296029652970Ile Glu Gly Asp Lys Gly Lys Gln Glu Glu Leu Val Cys Pro Ile297529802985Ser Pro Glu Asp Thr Ala Tyr Ile Met Tyr Thr Ser Gly Ser Thr299029953000Gly Lys Pro Lys Gly Val Met Val Thr His Arg Asn Ile Val Arg300530103015Leu Val Lys Asn Thr Asn Phe Val Ser Leu Gln Glu Gln Asp Val302030253030Leu Leu Gln Thr Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu303530403045Ile Trp Gly Ala Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu305030553060Asp Tyr Val Ile Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln306530703075Gln Asn Lys Ala Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn308030853090Gln Leu Ala Asp Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln309531003105
Leu Leu Ile Gly Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys311031153120Val Met Asp His Cys Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly312531303135Pro Thr Glu Asn Thr Thr Phe Ser Thr Ser Phe Val Ile Asp Gln314031453150Met Tyr Gln Asp Ser Ile Pro Ile Gly Thr Pro Ile Ala Asn Ser315531603165Ser Ala Tyr Ile Leu Asp Val His Gln Asn Ile Gln Pro Ile Gly317031753180Val Val Gly Glu Leu Cys Val Gly Gly Asp Gly Val Ala Lys Gly318531903195Tyr Val Asn Leu Glu Gln Leu Thr Glu Glu Arg Phe Ile Ala Asp320032053210Pro Phe Leu Lys Gly Ser Thr Met Tyr Arg Thr Gly Asp Tyr Val321532203225Lys Leu Leu Pro Asn Gly Asn Ile Gln Tyr Ile Gly Arg Val Asp323032353240Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Glu Ala Ile324532503255Met Asn Thr Leu Lys Gln Cys Glu Ser Ile Lys Asp Val Ile Val326032653270
Val Val Gln Glu Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val327532803285Val Gly Glu Glu Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala329032953300Lys Lys His Leu Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe330533103315Ile Glu Glu Ile Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser332033253330Lys Leu Pro Lys Val Gln Glu Val Leu His Lys Lys Val Glu Thr333533403345Leu Leu Pro Glu Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr335033553360Arg Asp Val Leu Glu Lys Glu Asp Phe Gly Val Thr Asp Ser Phe336533703375Phe Ala Tyr Gly Gly Asp Ser Leu Leu Ser Ile Gln Val Val Ser338033853390Met Leu Lys Lys Glu Glu Ile Ala Val Asp Pro Lys Met Ile Phe339534003405Met His Thr Thr Val Arg Glu Leu Ala Lys Ala Cys Glu Asn Arg341034153420Pro Val Met Glu Glu Thr Lys Arg Thr Glu Lys Asp Tyr Leu Ile342534303435
Gln Met Arg Glu Gly Ser Glu Glu Asp Ser Cys Ile Ile Phe Ala344034453450Pro Pro Ala Gly Gly Thr Val Leu Gly Tyr Ile Glu Leu Ala Arg345534603465Tyr Phe Glu Gly Ile Gly Asn Val Tyr Gly Leu Gln Ala Pro Gly347034753480Leu Tyr Asp Asp Glu Glu Pro Thr Phe Leu Asp Tyr Asp Glu Leu348534903495Val Gln Val Phe Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro Gly350035053510Gln Asp Tyr Leu Gly Gly His Ser Leu Gly Gly His Ile Ala Phe351535203525Gly Met Cys Cys Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly353035353540Leu Leu Ile Leu Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly354535503555Ala Lys Asp Glu Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val356035653570Leu Ala Ala Gly Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu357535803585Leu Lys Gln Leu Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala359035953600
Val Ala Gln Lys Asp Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr360536103615Tyr Leu Asp Lys Tyr Leu Lys Leu Gln Ile His Ser Leu Leu Met362036253630Ser Arg Thr Leu Glu Leu Glu Lys Thr Gln Leu Asp Ile Pro Ile363536403645Lys Val Phe Lys Thr Gln Phe His Thr Glu Glu Leu Val Glu Arg365036553660Phe Asp Ala Trp His Asn Tyr Thr Asn Gln Ala Cys Thr Phe Ile366536703675Asp Ile Pro Gly Thr His Thr Thr Met Met Arg Leu Pro His Val368036853690Lys Glu Val Ala Lys Lys Ile Glu Glu Gln Leu3695370021022111804212PRT213蠟狀芽孢桿菌4002Phe Ile Lys Ser Met Asn Gln Leu Gly Lys Ser Lys Asn Leu His Asn1 5 10 15Gly Gly Met Met Glu Met Lys Arg Val Glu Glu His Asp His Ile His20 25 30Val Leu Asn Glu Ile Glu Asn Glu Cys Glu Arg Arg Tyr Gly Arg Ser
35 40 45Asn Ile Ala Ile Met Leu Glu Lys His Gly Val His Glu Gln Pro Leu50 55 60His Ile Glu Asp Leu Phe His Glu Val Glu Met Gln Glu His Ser Arg65 70 75 80Val Ser Arg His Glu Thr Val Leu Met Thr Asp Lys Gln Cys Ile Asp85 90 95Glu Ser Gly Lys Pro Leu Ala Leu Arg Phe Gly Glu Pro Leu His Leu100 105 110Asp Asp Cys Thr Pro Lys Thr Leu Gln Glu Ile Leu Lys Arg Ala Ala115 120 125Lys Gln Ala Lys Asp Lys Gly Met Thr Phe Val Tyr Glu Asp Gly His130 135 140Glu Glu Tyr Leu Ser Tyr Gln Glu Met Leu Ala Asp Ala Glu Arg Leu145 150 155 160Leu Lys Gly Leu Arg Asn Leu Gly Ile Gln Pro Gly Glu Ser Ile Leu165 170 175Phe Gln Phe Lys Asp Asn Lys His Phe Val Thr Ala Phe Trp Ala Cys180 185 190Ile Leu Gly Gly Phe Leu Pro Thr Pro Leu Gly Thr Ala Pro Ile Tyr195 200 205Ser Glu Gln Asn Ala Gln Val Leu Lys Leu Tyr Asn Thr Trp Gln Leu
210 215 220Leu Glu Gln Pro Ile Ile Leu Thr Glu Phe Glu Leu Lys Glu Glu Ile225 230 235 240Ala Ala Ile Arg Thr Thr Leu Gln Arg Gln Glu Ile Val Ile His Ser245 250 255Ile Glu Asn Val Met Asp Thr Ala Arg Asp Thr Asn Trp Phe Pro Cys260 265 270Thr Glu Asp Thr Ile Val Leu Asn Leu Leu Thr Ser Gly Ser Thr Gly275 280 285Val Pro Lys Cys Val Gln His Lys Ser Lys Ser Ile Ile Ala Arg Thr290 295 300Val Ser Asn Cys Ile Asp Arg Gln Leu Asp Glu Lys Glu Val Ser Leu305 310 315 320Asn Trp Met Pro Leu Asp His Val Gly Gly Ile Val Met Cys His Ile325 330 335Arg Asp Thr Tyr Leu Met Cys Gln Gln Val Asn Cys Leu Ile Ser Ala340 345 350Phe Ile Glu Asn Pro Leu Asn Trp Leu His Trp Ile Asp Ala Tyr Ser355 360 365Ala Thr Phe Thr Trp Ala Pro Asn Phe Ala Phe Ser Leu Ile Asn Gln370 375 380Tyr Glu Glu Glu Ile Lys Ser Ser Ser Trp Asn Leu Ser Ser Met Arg
385 390 395 400Tyr Ile Val Asn Gly Gly Glu Ala Val Ile Ser Ser Val Gly Met Lys405 410 415Phe Leu Gln Leu Leu Gln Gln His Gln Leu Pro Ser Asn Cys Leu Ile420 425 430Pro Thr Phe Gly Met Ser Glu Val Ser Ser Gly Ile Ile Glu Cys His435 440 445Ser Phe Tyr Thr Gln Thr Thr Asn Thr Gly Met Leu Tyr Val Asp Lys450 455 460Asn Ser Leu Asp Gly Asn Leu Gln Phe Thr Tyr Glu Gly His Gln Asn465 470 475 480Ala Ile Val Phe Thr Glu Val Gly Arg Pro Met Pro Gly Ile Gly Ile485 490 495Arg Ile Val Asp Glu Asp Asn Gln Cys Leu Ser Glu Asp Arg Ile Gly500 505 510Arg Phe Gln Ile His Gly Pro Thr Val Met Asn Gly Tyr Phe Lys Asn515 520 525Asp Glu Ala Asn Ala Glu Ser Phe Thr Glu Asp Gly Trp Phe Asp Ser530 535 540Gly Asp Leu Gly Phe Ile His Asn Gly Asn Leu Val Ile Thr Gly Arg545 550 555 560Lys Lys Asp Met Ile Val Val His Gly Ala Asn Tyr Tyr Asn Tyr Glu
565 570 575Ile Glu Ala Leu Val Glu Gln Val Pro Gly Val Glu Thr Thr Phe Val580 585 590Cys Ala Thr Ser Val Lys Ser Ala Glu Gly Ala Glu Glu Leu Ala Ile595 600 605Phe Phe Val Pro Val Ile Asn His Val Ser Val Met Phe Ala Thr Met610 615 620Gln Gln Ile Lys Gln Ile Val Ala Arg Lys Met Gly Ile Thr Pro Lys625 630 635 640Val Ile Ile Pro Ile Gln Lys Glu Ala Phe Phe Lys Thr Asp Ser Gly645 650 655Lys Ile Thr Arg Asn Ala Phe Gln Lys Gln Phe Glu Asn Gly Ala Tyr660 665 670Arg Glu Ile Thr Gln Lys Ile Asp Cys His Leu Gln Asn Glu Lys Thr675 680 685Leu Ser Gln Trp Phe Tyr Arg Glu Lys Leu Val Glu Ser Lys Leu Gly690 695 700Lys Ser Val Ser Ser Gln Lys Glu Thr Tyr Val Phe Phe Arg Gln Gly705 710 715 720Lys Ser Phe His His Val Leu Lys Glu Lys Leu Thr Gln His Ser Val725 730 735Val Ile Val Asp Val Gly Glu Thr Phe Gly Glu Ile His Pro Asn His
740 745 750Tyr Gln Ile Asn Pro Lys Asn Lys Met Asp Tyr Val Arg Leu Phe Glu755 760 765Glu Leu Ala Lys Arg Asn Val Glu Asp Gln Val Phe His Leu Leu His770 775 780Ala Trp Asn Tyr Cys Asp Thr Val Pro Thr Phe Arg Ser Val Glu Asp785 790 795 800Leu Ala Asn Ala Gln Tyr Leu Gly Val Phe Ser Val Met Phe Ala Leu805 810 815Gln Ala Ile Met His Ala Lys Leu Pro Leu Arg Arg Val Thr Val Ile820 825 830Ala Thr Asn Ser Val Gly Leu Glu Ala Lys Glu Met Asn Tyr Ser Cys835 840 845Ser Thr Leu Glu Gly Tyr Val Lys Thr Leu Pro Ala Glu Phe Glu Asn850 855 860Leu Gln Val Lys Tyr Ile Asp Ile Glu Gly Lys Asp Ile Gln Phe Asp865 870 875 880Thr Glu Thr Val Trp Lys Glu Leu Gln Gln Gln Glu Thr Ile Pro Val885 890 895Val Leu Tyr Arg Asp Glu Lys Arg Tyr Lys Ile Gly Leu Glu Lys Val900 905 910Pro Met Leu Glu Gln Lys Glu Lys Asn Ile Pro Phe Gln Gln Gln Gly
915 920 925Phe Tyr Ile Ile Thr Gly Gly Leu Gly Gly Leu Gly Thr Leu Val Ala930 935 940Lys Leu Leu Leu Glu Arg Tyr Ser Ala Asn Val Leu Leu Leu Gly Arg945 950 955 960Thr Glu Ile Glu Thr Asn Ala Glu Lys Met Arg Leu Leu Asp Ser Leu965 970 975Lys Glu Tyr Glu Gln Tyr Gly Gly Thr Val Gln Tyr Lys Met Cys Asn980 985 990Val Met Asp Leu Asp Ala Met Arg Lys Val Val His Ser Gln Glu Glu995 10001005Arg Leu Gln Gln Lys Val Asn Gly Ile Ile His Leu Ala Gly Ile101010151020Ile Gln Glu Ile Leu Ile Glu Lys Gln Thr Glu Lys Glu Leu His102510301035Ala Met Phe Glu Ala Lys Val Tyr Ala Ser Trp Val Leu His Glu104010451050Ile Val Lys Glu Arg Gln Asp Cys Leu Tyr Ile Thr Thr Ser Ser105510601065Ala Arg Thr Leu Leu Pro Gly Met Thr Ile Ser Ala Tyr Cys Ser107010751080Ala Asn Arg Phe Val Glu Asn Phe Ala Tyr Tyr Gln Arg Ser Gln
108510901095Asn Val Asn Ser Tyr Cys Phe Ser Trp Ser Phe Trp Asn Glu Ile110011051110Gly Met Gly Thr Asn Leu Leu Ile Lys Asn Ala Leu Ile Ala Lys111511201125Gly Phe Gln Leu Ile Asp Asp Gln Lys Gly Ile Tyr Ser Leu Leu113011351140Ala Gly Leu Lys Gly Asn Glu Pro Asn Val Phe Val Gly Ile Asn114511501155His Glu Lys Glu Glu Met Ala His Leu Ile Gly Thr Glu Glu Gln116011651170Glu Thr Gln Gln Leu Thr Ile Tyr Ile Thr Pro Glu Tyr Leu His117511801185Ile Leu Glu Glu Val Phe Ser Ile Leu Asn Arg Glu Glu Phe Gly119011951200Gly Leu Glu Lys Glu Ile Val Ile Leu Pro Lys Leu Pro Leu Asp120512101215Glu Tyr Gly Lys Val Asp Gln Thr Arg Leu Ala His Ala Ser Asp122012251230Ser Arg Phe Gly Lys Lys Gln His Ile Val Pro Arg Asn Asp Ile123512401245Glu Glu Lys Ile Ala Phe Ile Trp Glu Gly Leu Leu Asn Lys Lys
125012551260Asp Ile Ser Val Leu Asp His Phe Phe Glu Leu Gly Gly Asp Ser126512701275Leu Lys Ala Thr Gln Met Ile Ser Ala Leu Lys Lys Asn Phe Ala128012851290Val Thr Ile Thr Gln Gln Glu Phe Phe Gln Ser Ser Thr Val Glu129513001305Glu Leu Ala Ser Leu Val Glu Lys Lys Leu Ser Arg Thr Arg Thr131013151320His Glu Met Asp Ile Val Thr Phe Ser Asp Arg Gly Asn Val Val132513301335Glu Met Ser Ser Ala Gln Lys Arg Gln Trp Phe Leu Tyr Glu Met134013451350Asp Arg Glu Asn Pro Tyr Tyr Asn Asn Thr Leu Val Ile Arg Leu135513601365Thr Gly Glu Ile His Leu Pro Ile Leu Arg Ser Ser Ile Ile Glu137013751380Leu Val Asn Lys His Glu Thr Leu Arg Thr Thr Phe Val Met Val138513901395Asp Gly Ile Pro Ser Gln Ile Ile Ala Asp Glu Glu Leu Val Glu140014051410Ile Glu Glu Ile Asp Leu Lys His Leu Ser Ala Glu Glu Thr Leu
141514201425Gln Lys Leu Glu Gly Leu Arg Gln Arg Glu Ala Asn Thr Ala Phe143014351440Lys Ile Glu Asn Ser Ala Phe Arg Ala Lys Val Ile Leu Ile Asp144514501455Glu Lys Arg Val Glu Ile Leu Leu Ser Val His His Ile Val Ser146014651470Asp Gly Trp Ser Met Gly Ile Leu Val Lys Asp Ile Ala Glu Ile147514801485Tyr Glu Asp Ile Arg Gln Trp Gly Glu Ser Lys Gln Glu Pro Leu149014951500Pro Ile Gln Tyr Ala Asp Tyr Thr Leu Trp Gln Asn Glu Phe Met150515101515Lys Gly Glu Glu Phe Ser Lys Gln Leu Ser Tyr Trp Lys Glu Lys152015251530Leu Ala Glu Asp Ile Pro Val Leu Asp Leu Pro Leu Asp Lys Pro153515401545Arg Pro Pro Ile Gln Thr Tyr Arg Gly Lys Val Lys Thr Phe Thr155015551560Leu His Glu Asn Met Thr Arg Met Leu Lys Glu Ile Cys Gln Glu156515701575Glu Glu Cys Thr Leu Phe Met Leu Leu Leu Ser Ala Phe Ser Ser
158015851590Leu Leu His Arg Tyr Thr Gly Gln Glu Asp Leu Val Val Gly Ser159516001605Leu Val Ala Asn Arg Asn Arg Glu Gln Ile Glu Lys Leu Ile Gly161016151620Phe Phe Val Asn Thr Leu Pro Leu Arg Ile Asn Leu His Arg Glu162516301635Met Gln Phe Thr Glu Leu Leu Ser Gln Val Lys Lys Thr Thr Ile164016451650Asp Ala Tyr Asp His Gln Asp Val Pro Phe Glu Leu Leu Val Asp165516601665Glu Leu Gln Ile Glu Arg Asp Ser Ser Arg Asn Ala Leu Phe Gln167016751680Val Leu Phe Val Leu Gln Asn Ala Gln Leu Gln Ala Val Asp Leu168516901695Glu Lys Ala Thr Met Glu Leu Glu Ile Leu Asp Ser Asp Thr Ala170017051710Lys Phe Asp Met Ser Val Gln Ile Phe Glu Leu Glu Asp Thr Leu171517201725Ser Ile Lys Leu Glu Tyr Asn Thr Asp Leu Phe Phe Asp Asp Thr173017351740Ile Glu Arg Phe Leu Ala His Tyr Glu Thr Ile Leu Ala Ser Val
174517501755Ile His Asn Gln Lys Ala Lys Ile Gly Glu Leu Ser Ile Leu Pro176017651770Gln Ser Glu Tyr Thr Lys Leu Val Ser Glu Trp Asn Glu Lys Ser177517801785Ala Thr Tyr Asn Gly Asn Gln Cys Ile His Glu Leu Phe Glu Ala179017951800Ala21032111900212PRT213蠟狀芽孢桿菌4003Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn Lys Glu1 5 10 15Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu Ala His Lys20 25 30Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val Gly Val Leu Cys35 40 45Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu Ala Val Leu Lys Ala50 55 60Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala Tyr Pro Met Gln Arg Thr65 70 75 80
Glu Tyr Val Leu Gln Asn Ser Glu Ala Thr Ile Leu Leu Thr Lys Glu85 90 95Cys Tyr Leu Lys Glu Ser Leu Asp Phe Glu Gly Glu Val Phe Tyr Leu100 105 110Asp Asp Ala Arg Leu Phe Glu Gly Asp Arg Arg Asp Leu Gln Asn Ile115 120 125Asn Asn Pro Thr Asn Leu Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr130 135 140Gly Asn Pro Lys Gly Val Met Val Ala His Gln Ser Val Val Asn Leu145 150 155 160Leu Leu Asp Leu Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His165 170 175Leu Leu Lys Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe180 185 190Gly Trp Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp195 200 205Glu Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val210 215 220Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln Ala225 230 235 240Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr Ile Ile245 250 255
Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg Leu Tyr Ala260 265 270His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly Pro Thr Glu Gly275 280 285Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys Glu Met Asn Val Ala290 295 300Asn Val Pro Ile Gly Lys Pro Leu Ser His Val Glu Thr Tyr Ile Leu305 310 315 320Asp Gln Asn Asn Gln Ile Val Pro Ile Gly Val Pro Gly Glu Leu Cys325 330 335Leu Gly Gly Ile Cys Val Ala Lys Gly Tyr Met Lys Glu Pro Val Leu340 345 350Thr Glu Glu Lys Phe Val Val Asn Pro Met Lys Gln Ser Glu Arg Met355 360 365Tyr Arg Thr Gly Asp Leu Val Arg Trp Leu Ala Asp Gly Asn Ile Glu370 375 380Tyr Leu Gly Arg Ile Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile385 390 395 400Glu Leu Gly Glu Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val405 410 415Gln Thr Ile Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile420 425 430
Val Ala Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu435 440 445His Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val450 455 460Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg Lys465 470 475 480Gln Leu His Ser Val Asp Leu Tyr Glu Thr Ser Met Asp Thr Val Ile485 490 495Val Gly Pro Arg Asn Glu Lys Glu Ala Met Leu Ser Val Ile Trp Gln500 505 510Glu Leu Leu Gly Leu Glu Asn Ile Ser Val His Asp Asn Phe Phe Lys515 520 525Leu Gly Gly His Ser Ile Asn Ala Thr Gln Leu Val Ser Lys Ile Tyr530 535 540Ser Val Cys Arg Val Arg Met Pro Leu Lys Asn Val Phe Gln Tyr Thr545 550 555 560Thr Leu Ala Thr Met Ala Arg Val Leu Glu Glu Leu Leu Val Ser Ala565 570 575Val Asp Glu Val Ala Val Thr Thr Glu Arg Ile Pro Lys Ile Leu Pro580 585 590Arg Thr Tyr Tyr Asp Leu Ser Tyr Ser Gln Gln Arg Ile Tyr Phe Leu595 600 605
Ser Thr Met Glu Lys Glu Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp610 615 620Asp Ile Tyr Gly Lys Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln625 630 635 640Leu Leu Met Lys Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val645 650 655Asp Gly Lys Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val660 665 670Gln Phe Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu675 680 685Val Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala690 695 700Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly Glu705 710 715 720His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly Trp Ser725 730 735Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp Ala Phe Val740 745 750Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile Ser Ile Met Asp755 760 765Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu Asp Gln Asp Asp Arg770 775 780
Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu Leu Pro Thr Leu Glu Leu785 790 795 800Pro Thr Asp Arg Gln Arg Pro Leu Leu Lys Thr Tyr His Gly Asp Thr805 810 815Leu Ser Tyr Lys Val Asn Ser Gln Leu His Gln Lys Leu Lys Asp Phe820 825 830Ser His Ala Asn Gly Val Thr Met Phe Met Thr Leu Leu Thr Ala Tyr835 840 845Asn Ile Met Leu Asn Lys Leu Thr Asn Glu Thr Asp Ile Val Val Gly850 855 860Ser Pro Val Ala Gly Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly865 870 875 880Met Phe Val Asn Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro885 890 895Thr Val Asp Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala900 905 910Tyr Asn His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp915 920 925Pro His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly930 935 940Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val Arg945 950 955 960
Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr Leu His965 970 975Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu Tyr Asn Thr980 985 990Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln Asn His Phe Glu995 10001005Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr Lys Arg Ile Ser101010151020Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr Glu Ile Leu Glu102510301035Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His Glu Ser Val Ala104010451050Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln Ala Lys Leu Ala105510601065Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala Glu Leu Ser Glu107010751080Lys Ala Asn Ala Leu Ala His Thr Leu Lys Arg Arg Gly Val Ala108510901095Gln His Asp Val Val Gly Ile Val Ala Glu Arg Ser Pro Glu Thr110011051110Ile Ile Gly Ile Leu Ala Ile Leu Lys Val Gly Ala Ile Tyr Leu111511201125
Pro Ile Asp Pro Lys Leu Pro Gln Leu Thr Leu Gln His Ile Trp113011351140Arg Asp Ser Gly Ala Lys Val Leu Leu Gly Lys Asn Glu Thr Thr114511501155Val Glu Val Gly Lys Glu Val Pro Phe Val Asp Ile Glu Gly Asp116011651170Lys Gly Lys Gln Glu Glu Leu Val Cys Pro Ile Ser Pro Glu Asp117511801185Thr Ala Tyr Ile Met Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys119011951200Gly Val Met Val Thr His Arg Asn Ile Val Arg Leu Val Lys Asn120512101215Thr Asn Phe Val Ser Leu Gln Glu Gln Asp Val Leu Leu Gln Thr122012251230Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu Ile Trp Gly Ala123512401245Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu Asp Tyr Val Ile125012551260Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln Gln Asn Lys Ala126512701275Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn Gln Leu Ala Asp128012851290
Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln Leu Leu Ile Gly129513001305Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys Val Met Asp His131013151320Cys Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly Pro Thr Glu Asn132513301335Thr Thr Phe Ser Thr Ser Phe Val Ile Asp Gln Met Tyr Gln Asp134013451350Ser Ile Pro Ile Gly Thr Pro Ile Ala Asn Ser Ser Ala Tyr Ile135513601365Leu Asp Val His Gln Asn Ile Gln Pro Ile Gly Val Val Gly Glu137013751380Leu Cys Val Gly Gly Asp Gly Val Ala Lys Gly Tyr Val Asn Leu138513901395Glu Gln Leu Thr Glu Glu Arg Phe Ile Ala Asp Pro Phe Leu Lys140014051410Gly Ser Thr Met Tyr Arg Thr Gly Asp Tyr Val Lys Leu Leu Pro141514201425Asn Gly Asn Ile Gln Tyr Ile Gly Arg Val Asp Asn Gln Val Lys143014351440Ile Arg Gly Phe Arg Ile Glu Leu Glu Ala Ile Met Asn Thr Leu144514501455
Lys Gln Cys Glu Ser Ile Lys Asp Val Ile Val Val Val Gln Glu146014651470Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val Val Gly Glu Glu147514801485Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala Lys Lys His Leu149014951500Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe Ile Glu Glu Ile150515101515Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser Lys Leu Pro Lys152015251530Val Gln Glu Val Leu His Lys Lys Val Glu Thr Leu Leu Pro Glu153515401545Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr Arg Asp Val Leu155015551560Glu Lys Glu Asp Phe Gly Val Thr Asp Ser Phe Phe Ala Tyr Gly156515701575Gly Asp Ser Leu Leu Ser Ile Gln Val Val Ser Met Leu Lys Lys158015851590Glu Glu Ile Ala Val Asp Pro Lys Met Ile Phe Met His Thr Thr159516001605Val Arg Glu Leu Ala Lys Ala Cys Glu Asn Arg Pro Val Met Glu161016151620
Glu Thr Lys Arg Thr Glu Lys Asp Tyr Leu Ile Gln Met Arg Glu162516301635Gly Ser Glu Glu Asp Ser Cys Ile Ile Phe Ala Pro Pro Ala Gly164016451650Gly Thr Val Leu Gly Tyr Ile Glu Leu Ala Arg Tyr Phe Glu Gly165516601665Ile Gly Asn Val Tyr Gly Leu Gln Ala Pro Gly Leu Tyr Asp Asp167016751680Glu Glu Pro Thr Phe Leu Asp Tyr Asp Glu Leu Val Gln Val Phe168516901695Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro Gly Gln Asp Tyr Leu170017051710Gly Gly His Ser Leu Gly Gly His Ile Ala Phe Gly Met Cys Cys171517201725Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly Leu Leu Ile Leu173017351740Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly Ala Lys Asp Glu174517501755Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val Leu Ala Ala Gly176017651770Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu Leu Lys Gln Leu177517801785
Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala Val Ala Gln Lys179017951800Asp Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr Tyr Leu Asp Lys180518101815Tyr Leu Lys Leu Gln Ile His Ser Leu Leu Met Ser Arg Thr Leu182018251830Glu Leu Glu Lys Thr Gln Leu Asp Ile Pro Ile Lys Val Phe Lys183518401845Thr Gln Phe His Thr Glu Glu Leu Val Glu Arg Phe Asp Ala Trp185018551860His Asn Tyr Thr Asn Gln Ala Cys Thr Phe Ile Asp Ile Pro Gly186518701875Thr His Thr Thr Met Met Arg Leu Pro His Val Lys Glu Val Ala188018851890Lys Lys Ile Glu Glu Gln Leu1895190021042111020212PRT213蠟狀芽孢桿菌4004Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn Lys Glu1 5 10 15
Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu Ala His Lys20 25 30Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val Gly Val Leu Cys35 40 45Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu Ala Val Leu Lys Ala50 55 60Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala Tyr Pro Met Gln Arg Thr65 70 75 80Glu Tyr Val Leu Gln Asn Ser Glu Ala Thr Ile Leu Leu Thr Lys Glu85 90 95Cys Tyr Leu Lys Glu Ser Leu Asp Phe Glu Gly Glu Val Phe Tyr Leu100 105 110Asp Asp Ala Arg Leu Phe Glu Gly Asp Arg Arg Asp Leu Gln Asn Ile115 120 125Asn Asn Pro Thr Asn Leu Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr130 135 140Gly Asn Pro Lys Gly Val Met Val Ala His Gln Ser Val Val Asn Leu145 150 155 160Leu Leu Asp Leu Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His165 170 175Leu Leu Lys Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe180 185 190
Gly Trp Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp195 200 205Glu Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val210 215 220Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln Ala225 230 235 240Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr Ile Ile245 250 255Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg Leu Tyr Ala260 265 270His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly Pro Thr Glu Gly275 280 285Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys Glu Met Asn Val Ala290 295 300Asn Val Pro Ile Gly Lys Pro Leu Ser His Val Glu Thr Tyr Ile Leu305 310 315 320Asp Gln Asn Asn Gln Ile Val Pro Ile Gly Val Pro Gly Glu Leu Cys325 330 335Leu Gly Gly Ile Cys Val Ala Lys Gly Tyr Met Lys Glu Pro Val Leu340 345 350Thr Glu Glu Lys Phe Val Val Asn Pro Met Lys Gln Ser Glu Arg Met355 360 365
Tyr Arg Thr Gly Asp Leu Val Arg Trp Leu Ala Asp Gly Asn Ile Glu370 375 380Tyr Leu Gly Arg Ile Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile385390 395 400Glu Leu Gly Glu Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val405 410 415Gln Thr Ile Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile420 425 430Val Ala Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu435 440 445His Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val450 455 460Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg Lys465 470 475 480Gln Leu His Ser Val Asp Leu Tyr Glu Thr Ser Met Asp Thr Val Ile485 490 495Val Gly Pro Arg Asn Glu Lys Glu Ala Met Leu Ser Val Ile Trp Gln500 505 510Glu Leu Leu Gly Leu Glu Asn Ile Ser Val His Asp Asn Phe Phe Lys515 520 525Leu Gly Gly His Ser Ile Asn Ala Thr Gln Leu Val Ser Lys Ile Tyr530 535 540
Ser Val Cys Arg Val Arg Met Pro Leu Lys Asn Val Phe Gln Tyr Thr545 550 555 560Thr Leu Ala Thr Met Ala Arg Val Leu Glu Glu Leu Leu Val Ser Ala565 570 575Val Asp Glu Val Ala Val Thr Thr Glu Arg Ile Pro Lys Ile Leu Pro580 585 590Arg Thr Tyr Tyr Asp Leu Ser Tyr Ser Gln Gln Arg Ile Tyr Phe Leu595 600 605Ser Thr Met Glu Lys Glu Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp610 615 620Asp Ile Tyr Gly Lys Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln625 630 635 640Leu Leu Met Lys Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val645 650 655Asp Gly Lys Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val660 665 670Gln Phe Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu675 680 685Val Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala690 695 700Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly Glu705 710 715 720
His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly Trp Ser725 730 735Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp Ala Phe Val740 745 750Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile Ser Ile Met Asp755 760 765Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu Asp Gln Asp Asp Arg770 775 780Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu Leu Pro Thr Leu Glu Leu785 790 795 800Pro Thr Asp Arg Gln Arg Pro Leu Leu Lys Thr Tyr His Gly Asp Thr805 810 815Leu Ser Tyr Lys Val Asn Ser Gln Leu His Gln Lys Leu Lys Asp Phe820 825 830Ser His Ala Asn Gly Val Thr Met Phe Met Thr Leu Leu Thr Ala Tyr835 840 845Asn Ile Met Leu Asn Lys Leu Thr Asn Glu Thr Asp Ile Val Val Gly850 855 860Ser Pro Val Ala Gly Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly865 870 875 880Met Phe Val Asn Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro885 890 895
Thr Val Asp Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala900 905 910Tyr Asn His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp915 920 925Pro His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly930 935 940Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val Arg945 950 955 960Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr Leu His965 970 975Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu Tyr Asn Thr980 985 990Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln Asn His Phe Glu995 10001005Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr Lys1010101510202105211880212PRT213蠟狀芽孢桿菌4005Arg Ile Ser Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr Glu Ile1 5 10 15
Leu Glu Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His Glu Ser Val20 25 30Ala Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln Ala Lys Leu Ala35 40 45Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala Glu Leu Ser Glu Lys50 55 60Ala Asn Ala Leu Ala His Thr Leu Lys Arg Arg Gly Val Ala Gln His65 70 75 80Asp Val Val Gly Ile Val Ala Glu Arg Ser Pro Glu Thr Ile Ile Gly85 90 95Ile Leu Ala Ile Leu Lys Val Gly Ala Ile Tyr Leu Pro Ile Asp Pro100 105 110Lys Leu Pro Gln Leu Thr Leu Gln His Ile Trp Arg Asp Ser Gly Ala115 120 125Lys Val Leu Leu Gly Lys Asn Glu Thr Thr Val Glu Val Gly Lys Glu130 135 140Val Pro Phe Val Asp Ile Glu Gly Asp Lys Gly Lys Gln Glu Glu Leu145 150 155 160Val Cys Pro Ile Ser Pro Glu Asp Thr Ala Tyr Ile Met Tyr Thr Ser165 170 175Gly Ser Thr Gly Lys Pro Lys Gly Val Met Val Thr His Arg Asn Ile180 185 190
Val Arg Leu Val Lys Asn Thr Asn Phe Val Ser Leu Gln Glu Gln Asp195 200 205Val Leu Leu Gln Thr Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu210 215 220Ile Trp Gly Ala Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu Asp225 230 235 240Tyr Val Ile Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln Gln Asn245 250 255Lys Ala Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn Gln Leu Ala260 265 270Asp Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln Leu Leu Ile Gly275 280 285Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys Val Met Asp His Cys290 295 300Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly Pro Thr Glu Asn Thr Thr305 310 315 320Phe Ser Thr Ser Phe Val Ile Asp Gln Met Tyr Gln Asp Ser Ile Pro325 330 335Ile Gly Thr Pro Ile Ala Asn Ser Ser Ala Tyr Ile Leu Asp Val His340 345 350Gln Asn Ile Gln Pro Ile Gly Val Val Gly Glu Leu Cys Val Gly Gly355 360 365
Asp Gly Val Ala Lys Gly Tyr Val Asn Leu Glu Gln Leu Thr Glu Glu370 375 380Arg Phe Ile Ala Asp Pro Phe Leu Lys Gly Ser Thr Met Tyr Arg Thr385 390 395 400Gly Asp Tyr Val Lys Leu Leu Pro Asn Gly Asn Ile Gln Tyr Ile Gly405 410 415Arg Val Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Glu420 425 430Ala Ile Met Asn Thr Leu Lys Gln Cys Glu Ser Ile Lys Asp Val Ile435 440 445Val Val Val Gln Glu Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val450 455 460Val Gly Glu Glu Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala Lys465 470 475 480Lys His Leu Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe Ile Glu485 490 495Glu Ile Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser Lys Leu Pro500 505 510Lys Val Gln Glu Val Leu His Lys Lys Val Glu Thr Leu Leu Pro Glu515 520 525Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr Arg Asp Val Leu Glu530 535 540
Lys Glu Asp Phe Gly Val Thr Asp Ser Phe Phe Ala Tyr Gly Gly Asp545 550 555 560Ser Leu Leu Ser Ile Gln Val Val Ser Met Leu Lys Lys Glu Glu Ile565 570 575Ala Val Asp Pro Lys Met Ile Phe Met His Thr Thr Val Arg Glu Leu580 585 590Ala Lys Ala Cys Glu Asn Arg Pro Val Met Glu Glu Thr Lys Arg Thr595 600 605Glu Lys Asp Tyr Leu Ile Gln Met Arg Glu Gly Ser Glu Glu Asp Ser610 615 620Cys Ile Ile Phe Ala Pro Pro Ala Gly Gly Thr Val Leu Gly Tyr Ile625 630 635 640Glu Leu Ala Arg Tyr Phe Glu Gly Ile Gly Asn Val Tyr Gly Leu Gln645 650 655Ala Pro Gly Leu Tyr Asp Asp Glu Glu Pro Thr Phe Leu Asp Tyr Asp660 665 670Glu Leu Val Gln Val Phe Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro675 680 685Gly Gln Asp Tyr Leu Gly Gly His Ser Leu Gly Gly His Ile Ala Phe690 695 700Gly Met Cys Cys Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly Leu705 710 715 720
Leu Ile Leu Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly Ala Lys725 730 735Asp Glu Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val Leu Ala Ala740 745 750Gly Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu Leu Lys Gln Leu755 760 765Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala Val Ala Gln Lys Asp770 775 780Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr Tyr Leu Asp Lys Tyr Leu785 790 795 800Lys Leu Gln Ile His Ser Leu Leu Met Ser Arg Thr Leu Glu Leu Glu805 810 815Lys Thr Gln Leu Asp Ile Pro Ile Lys Val Phe Lys Thr Gln Phe His820 825 830Thr Glu Glu Leu Val Glu Arg Phe Asp Ala Trp His Asn Tyr Thr Asn835 840 845Gln Ala Cys Thr Phe Ile Asp Ile Pro Gly Thr His Thr Thr Met Met850 855 860Arg Leu Pro His Val Lys Glu Val Ala Lys Lys Ile Glu Glu Gln Leu865 870 875 880210621111115212DNA213蠟狀芽孢桿菌
4006tttataaaat caatgaatca gttgggaaaa tcaaaaaatt tacataatgg ggggatgatg60gagatgaaac gagtggaaga acatgatcac attcatgtgt taaatgaaat agaaaacgaa120tgcgaaagaa gatatgggag aagtaatatt gcaattatgc ttgaaaagca tggtgttcat180gaacagccgc ttcatataga agacttattt catgaggtag agatgcaaga acattcacga240gtatcgcgcc acgaaacagt tttgatgaca gataaacaat gtatagatga gagtggaaaa300ccgttagctc ttcgttttgg tgagccactt catcttgatg actgtacccc aaaaacacta360caagaaattt taaagcgtgc cgctaagcaa gcaaaagata aagggatgac atttgtatat420gaagatggac atgaagagta cctctcctac caagagatgt tggcagatgc ggagcggtta480ctaaaagggt tgcgaaatct tggtatacaa ccaggagaaa gtattttgtt tcaatttaag540gacaataagc attttgttac tgcgttttgg gcatgtatac ttgggggatt tttaccaacg600ccgttaggaa cggcccctat ctatagtgag caaaatgcac aagtattaaa actttataac660acatggcagc tattagaaca accgattatt ttaacggaat ttgaattgaa agaagagatt720gctgcaattc gaacaacatt gcaacgtcaa gagattgtta tacatagtat tgagaatgtt780atggatacag cgcgcgatac aaactggttt ccttgtaccg aagatactat tgttttgaat840ttattaacgt ctggtagcac aggagtaccc aaatgtgtgc agcacaaaag taaatccatt900attgcgcgca cagtttccaa ttgtattgac cgccagctag atgaaaaaga agtatcgtta960aattggatgc cgcttgatca tgttggaggc atcgtaatgt gtcacattcg tgatacctat1020ttaatgtgcc aacaggtaaa ctgtcttatt tcggcattta ttgaaaatcc gctaaattgg1080ttgcactgga ttgatgctta ctcagcgaca tttacatggg cgccaaactt cgctttttca1140ttaattaacc agtatgaaga agagattaaa tcatcttcat ggaatctttc ttcgatgaga1200tacatcgtaa atggtgggga agctgttatt tcaagtgttg ggatgaaatt tttacaattg1260
ttacagcaac atcaattgcc ttcgaactgt cttattccta cgtttgggat gtctgaagtt1320tcttcgggta ttattgaatg tcattcgttt tatacgcaaa cgacaaatac aggaatgttg1380tatgttgata agaattcttt agatggtaat ttacaattca catatgaggg gcaccaaaat1440gccattgttt ttacggaagt agggagaccg atgcctggta ttggcattcg tattgttgat1500gaggacaatc agtgcctgtc agaagatcgt attggacgat tccaaattca tggtccaacg1560gttatgaatg gttatttcaa aaatgatgaa gcaaatgcgg aaagttttac tgaagatggc1620tggttcgata gtggagatct ggggtttata cataacggta atcttgtcat tacaggaaga1680aaaaaggata tgattgttgt tcatggtgca aattactaca actatgaaat tgaagccttg1740gtagaacaag tacctggggt ggaaaccacg tttgtatgtg caacgagtgt gaagtcggct1800gaaggagcag aggaattagc tattttcttt gtcccagtaa ttaatcatgt ttctgtgatg1860tttgcgacga tgcaacaaat caaacaaatt gttgcgcgca aaatgggtat cacgccgaaa1920gtgattatac caattcagaa ggaagcattc tttaaaacgg atagtgggaa aataacgaga1980aatgcatttc aaaaacagtt tgagaacggg gcatatagag agattacaca aaagattgat2040tgccatttac aaaatgaaaa aacactatct cagtggtttt atcgtgaaaa attagtcgaa2100agtaagttgg gcaaaagcgt atcctcccaa aaagaaacgt atgttttctt tcgacaaggt2160aaatcatttc atcatgtcct aaaagaaaag ttaacgcaac attctgttgt tattgtggat2220gtaggagaaa cattcggtga gatccatcca aatcattatc aaattaatcc taaaaacaaa2280atggattacg ttcgattatt tgaagaactc gcaaaaagaa atgtagaaga tcaagttttt2340catctcttgc atgcttggaa ttattgcgat acagttccaa cttttagatc ggtagaagat2400ttagctaatg cgcaatatct tggtgtgttt agtgtgatgt tcgcacttca agctattatg2460catgcgaaat tgccactacg tcgtgttacg gtgattgcga caaatagtgt tggattagaa2520gcgaaggaaa tgaactattc atgttcaaca ttagaaggtt atgtgaaaac tttgccagct2580
gagtttgaaa atttacaagt gaagtatatt gatatagaag gaaaagatat acagtttgat2640actgagaccg tatggaaaga acttcagcaa caagaaacca ttcctgtcgt attgtatcgt2700gatgagaaaa gatacaaaat aggtttagaa aaagtaccaa tgttagaaca gaaagaaaaa2760aatattccgt ttcaacagca agggttttac atcattacag gtggtcttgg tggtttgggg2820acgcttgtag ccaaattact tttagaacga tacagcgcaa atgttctttt acttggtcga2880acagaaattg aaacaaatgc agaaaaaatg cgccttcttg attcattaaa agagtatgaa2940caatatggtg gtacagtcca atataaaatg tgcaatgtaa tggatttaga tgcgatgcga3000aaagttgttc attcacagga agaacgtctg caacaaaagg taaatgggat tatccacctt3060gcagggatta ttcaagaaat actgatagaa aagcaaaccg aaaaagaact gcatgctatg3120tttgaagcta aggtatatgc atcttgggtg ctacatgaaa tcgtaaaaga aaggcaagat3180tgtctctaca ttacaacttc ttcagcaaga acgttgttac cggggatgac catctcagct3240tattgtagtg cgaatcgatt tgttgaaaat tttgcatatt atcaacgaag tcaaaatgta3300aatagctact gtttttcatg gagtttctgg aatgagattg gaatgggtac aaatttactt3360attaaaaatg cgttgatagc aaaaggattt caattgatcg atgatcaaaa aggtatatat3420tcccttttgg cgggattaaa agggaacgaa cctaatgttt ttgttggaat caatcatgaa3480aaagaagaaa tggctcatct gattggaacc gaggaacaag aaacacaaca attaacaatc3540tatattacac cagaatactt acatattctt gaagaagtgt tctctatact aaatagagaa3600gaatttggtg gattggagaa agagattgtc attctaccaa aattaccgct tgatgaatat3660ggtaaagtag atcaaactcg tttggctcat gcgtcggata gccgttttgg aaagaaacaa3720catatcgtac caagaaatga tatagaagag aaaattgcat tcatttggga aggtcttttg3780aataaaaagg atattagtgt acttgaccat ttcttcgaat taggtggtga ttctttaaaa3840gcgacacaaa tgatttctgc gttgaaaaag aattttgctg ttacgattac gcaacaggaa3900
ttttttcaat cgagtacagt agaagagctt gctagtttag tagaaaagaa actttctcgt3960actcgtacgc atgaaatgga catagttact tttagtgacc gaggtaacgt agtagagatg4020tcttctgcac aaaagcggca atggttttta tatgaaatgg atcgagaaaa tccttattac4080aataatacac ttgtaattcg tttgacggga gaaattcatc ttcctatttt aagaagttct4140attattgagt tagtaaataa gcatgaaaca ttgcgaacaa catttgtgat ggtggatggt4200ataccatcac aaattattgc agatgaagag ttagttgaaa tagaggaaat tgatttgaaa4260cacctatctg ctgaggagac gttgcaaaaa ctagagggtt tacgacaacg ggaagcaaat4320acggcgttta aaatcgaaaa tagcgctttt cgtgcaaaag tgattttaat tgatgagaag4380agagtggaga ttttactttc cgtgcatcac attgtttcgg atggttggtc gatggggatt4440ttagtgaagg acattgcgga aatctatgaa gatattcggc agtggggaga aagtaagcaa4500gagccattac cgattcaata cgcagattat actttgtggc aaaatgagtt tatgaaaggt4560gaggaattta gcaagcaact gtcttattgg aaggagaaat tagctgaaga tatacctgta4620cttgatcttc cgttagataa accacggcca ccaattcaaa catatcgtgg gaaggttaag4680actttcacgt tacatgaaaa catgacaagg atgctaaaag aaatatgtca agaagaagaa4740tgcacgctct ttatgttgtt actttcggct ttctcatcat tattacatcg ttatacaggt4800caggaggatc ttgttgttgg ttcgctagtt gcaaatcgaa accgtgagca aatcgagaaa4860ttgattggtt tctttgttaa tacgttaccg ctacgtatta atcttcatcg ggaaatgcaa4920tttactgaat tgctttcgca agtaaagaaa acgaccattg atgcatatga tcatcaagat4980gtgccttttg agctactagt cgatgaatta cagattgaga gagattcgag tcgtaatgcg5040ctattccaag tgttgtttgt cttacaaaac gcacaattac aagcagtaga cttagagaaa5100gcgacaatgg aactcgaaat tttagatagt gacacggcca agtttgatat gtcagtgcaa5160attttcgaat tggaggacac tttatctatc aaattagagt acaatacgga tttatttttt5220
gatgatacaa tagaacgctt tcttgctcat tatgaaacca tattagcaag cgttattcat5280aatcaaaagg caaaaatagg ggaattgtca attttaccac aatctgaata tacgaaactt5340gtatctgagt ggaatgaaaa gagtgccact tataatggaa atcagtgtat tcatgaattg5400ttcgaagcag ctgttcacaa aacgccatct gcaacagcgc ttatttatcg caacaaagag5460atgacatacg aggatgttaa tgcgcaggca aatgcacttg cacataaatt aagagatgca5520ggtgttggac caaaccaggt agttggcgtg ttatgtgatc gctctttcga gatggttgtt5580ggtatattag ctgttttaaa agcaggtggt gcgtatttgc caattgatac agcgtacccg5640atgcaacgaa cagaatacgt cctgcaaaat agtgaggcaa ctattctctt aacaaaggaa5700tgttacctta aggagtcttt agattttgag ggggaagttt tttacttaga tgatgcaaga5760ctgtttgaag gggatagaag agatttacaa aatatcaata atcctactaa ccttgcttat5820atcatttata catcaggatc cacgggaaat ccaaaaggtg ttatggtagc gcatcaaagt5880gttgtgaatt tgctactcga tttacaagag aaatatccgg tgctagcaga agataagcac5940ttgttaaaaa caacatatac gtttgatgtt tctgtagccg aaatttttgg atggtttcat6000gcaggtggca cacttgttat tgctggacat ggtgatgaaa aagacccaga gaaactgatt6060caattgattc aatgccacaa ggttacacat attaacttcg taccatcgat gctacatgca6120atgttacagg ccttggatga aaaagatttt gcaattatga atcggttgaa atatattatc6180gtcgcaggag aagctgtttc accagaactt tgtaatcgac tgtacgctca ttgtccaaat6240gtaaaactag aaaatctata tgggccaacg gaaggaacga tttatgcgac agggttttct6300attcataaag aaatgaatgt agctaatgta ccgattggaa aaccactttc tcatgtggaa6360acgtatattc ttgatcaaaa caatcaaatt gtaccaattg gtgtaccagg tgaattgtgt6420ctgggaggaa tatgtgtagc aaaaggttat atgaaagagc cggtgttaac agaagaaaaa6480ttcgtcgtca atcctatgaa acaaagtgaa agaatgtacc gaacgggtga tttggtacgc6540
tggttagcag atgggaatat tgaatattta ggaagaatag ataaccaagt caagataaga6600ggcttccgaa ttgagcttgg tgaaattgaa gcggcaattg ctgcattaga agatgtagta6660caaacaattg ttacaacaat gacggatcat aaaggtgcga acaagattgt cgcatatgtt6720gtgagcgaaa agtatgatga agaacgaatt cgtgaacatg tgaaaaagac gttgccgcaa6780tatatggtac caagttattt cgtttcgatg aaggcattgc ctcttaataa aaatggaaaa6840gttgatcgca aacagttgca ttcggttgat ctttatgaaa cgagtatgga tacagtcatt6900gtgggaccaa gaaacgaaaa agaagcaatg ctttcggtta tttggcaaga gcttttggga6960ttagagaata tcagtgttca cgataatttc tttaagcttg gtggtcattc cattaatgcg7020acacaattgg tatcaaaaat ttatagtgtt tgccgagtga gaatgcctct taaaaatgtg7080tttcagtata caacgttagc tacaatggca cgggtgttag aagagttgtt ggtaagcgct7140gttgacgaag tagctgtaac aacggagcgc attccaaaga tactaccgag aacatattac7200gatttgtcgt attcacaaca aagaatttat ttcttatcta caatggagaa agaaaccaat7260tactataata ttcttggtgc ttgggatatt tatgggaaac tagatgttac gctatttgaa7320aaagcaatcc aactattaat gaagaaacac cattcattac gtgcaacatt tgaaatcgtg7380gacggcaaac ctgtgcaaat catccacgat gatatggaaa ttcctgtgca atttattgac7440cttactgtga tgccagaagg attacggata gaagaagtag atgaacttat gttaaaagag7500tctaaaagag tatacaatct cgcaaatggt ccgttaatgc attgtacaat tgttaagata7560aaagaaggtg agcatgtatt attgattgga caacatcata tcattagtga tggttggtca7620cttggtattt ttgtaaaaga gttaaatgaa atgtatgatg cctttgtgca acacaaacca7680gttgctgaaa caccatcaac aatctccatt atggacttta ctgcttggca caatagtaaa7740gtagatgaag atcaagatga tcgacaatat tggttacagc gatttgaggg agagttaccg7800acgttagagt tgccgacaga cagacaacgt ccacttttga aaacatatca tggtgacaca7860
ttatcatata aggtgaattc tcaattgcat caaaaattaa aggactttag tcatgcaaat7920ggtgtaacga tgtttatgac gctattaacg gcgtataata ttatgttgaa taagttaaca7980aatgaaacag acattgttgt tggctcccct gtagcaggta gaaatgaacc agaatcaaaa8040gatttaatcg ggatgtttgt gaatacgtta gcgttacgtt cgcatttagg agataatccg8100acagttgatg tcttattaaa acaaataaaa cagaatactt tagaagcata caatcatcaa8160gattatccat ttgataagtt ggttgatgac ttggatccac atcgagattt aagtaggaca8220ccaattttcc aagtgatgat gggatatatg aatatgccat tgatggttgc atttcgtgaa8280gcagaggttc gcgaacgatt tgttcgacat aaagtcgcaa ggtttgattt aacacttcat8340gtgtttgaag atgaagatca gatgaaaata ttctttgagt ataatacaga tttatttgat8400gaatcaacga ttatgcgttg gcagaatcat ttcgaaacgc tattacagga aattgtatcg8460aatccgacaa aacgtatttc ggaattgaat atacttacaa atgaggagaa atatgaaatt8520ctagagatga acaataattc aacggagtat cctcagcatg aatctgttgc ggagattttt8580agagaaacga agataaagca tcaagcaaaa ctagcaatta cgtacaaaga tagaaagtta8640acgtatgcag agttgagtga aaaagcaaat gcgttggcac atacattgaa acgtcgaggt8700gttgcgcagc atgatgttgt tggaattgtc gcagagcgtt cgcctgaaac aattattgga8760atactcgcaa tcttaaaagt aggagcaatt tatttgccaa ttgatccaaa actaccgcaa8820ttaacactgc aacacatttg gcgagatagc ggtgcaaaag tcctcctagg gaaaaatgaa8880acaactgtag aagttggcaa ggaagttccg tttgtggaca tcgaagggga taaagggaag8940caagaggagt tagtgtgtcc aattagtcca gaagatacgg catatattat gtatacgtca9000ggcagtactg gaaaaccaaa aggggttatg gtgacacata gaaatattgt tcgtttagta9060aaaaatacga atttcgtttc tttgcaagag caagatgtgt tgttacagac aggttcgctt9120acttttgacg ctgcaacatt tgaaatttgg ggcgcattgc taaatggact tacgcttcat9180
ttagtagaag attacgtaat tttagatggg gaggcgcttc aggaagagat tcagcagaac9240aaagcaacca ttatgtgggt gagtgcaccg ctgtttaatc aattggcgga tcaaaaccca9300gcgatgttta caggcattaa gcaattgctc attggtggtg atgttttatc gccaaaacat9360attaacaaag tgatggacca ttgtgcacca atcaatatca ttaatggata cggtccaaca9420gaaaatacga cgttctcgac gtcatttgta attgatcaaa tgtatcaaga cagcattccg9480attggaacac cgattgctaa ttctagtgct tacattttag atgtacatca aaatatacaa9540cctattggtg tagttggcga actatgtgtt ggtggtgatg gagttgcaaa aggttatgtg9600aaccttgaac aattaacaga agaacggttt atagcagatc cgttcctaaa gggttctaca9660atgtacagaa ccggcgatta tgtgaaatta ttgcctaatg gaaatattca atacattgga9720cgtgtggaca atcaagtgaa aattcgtgga ttccgcatcg aattagaagc cattatgaac9780acattaaaac aatgtgaatc aatcaaagat gtaattgttg ttgtacaaga acagaatggg9840tataaaacac tggttgcata tgttgtggga gaagaatcgc tttcaataga aacagtgagg9900gcctatgcaa aaaaacattt ggctgaatat atggtacctt ctcaatttat atttatagaa9960gaaattccgc tctcaataaa tgggaaagta cagtatagta agttaccgaa agtacaagaa10020gtattgcata aaaaagtaga aacgctgtta ccagaaaaca gattagaaga aattattcta10080cgtgtgtatc gtgatgtatt agagaaagaa gattttggcg taacagattc attcttcgct10140tatggtggtg actctttact aagtattcaa gtcgtttcga tgttgaaaaa agaggagatt10200gcagtagatc cgaaaatgat ttttatgcat acaacggtta gagagttagc aaaggcatgt10260gaaaatcgtc cggttatgga agaaacaaaa aggactgaga aggattattt aattcaaatg10320cgtgaaggta gtgaagaaga tagttgtatc atttttgctc ctccggcagg tggaacggta10380cttggatata tagaattagc aaggtatttc gagggaattg gcaatgttta cggcctacaa10440gcaccgggac tgtatgacga tgaagagcct acgttcttag attacgatga acttgtacaa10500
gtgtttcttc gctcgattga agggacatat cgtccaggtc aagattattt aggtggccac10560tccttagggg gacatatcgc atttggaatg tgctgtgaac tgattaagca aggaaaggca10620ccaaagggat tgctaattct agatacaaca ccatcacttc aagttgtaaa gggggccaag10680gatgaaaaaa tagccgagga ggactttaaa atgatggtac tggctgccgg tatcggaaat10740atgatgggtg ttgatccaga agaattaaag caactgtcgt atgaagaagc aaaaacaaga10800gttgtcgcag tggcacaaaa ggatgaaaag ttaaaaactt ttataaatga aacatatttg10860gataagtatt tgaagttaca aattcatagt ttactaatgt cacgaacatt agaattggag10920aaaacacaat tagatattcc gattaaggta tttaaaacac agtttcatac agaagagcta10980gtagaaagat ttgatgcttg gcataactat acaaatcaag cctgcacatt cattgatata11040ccaggcacac atacgacgat gatgcgttta ccacatgtga aagaggtagc gaaaaaaata11100gaagaacagc tataa 1111521072115700212DNA213蠟狀芽孢桿菌4007cacaaaacgc catctgcaac agcgcttatt tatcgcaaca aagagatgac atacgaggat60gttaatgcgc aggcaaatgc acttgcacat aaattaagag atgcaggtgt tggaccaaac120caggtagttg gcgtgttatg tgatcgctct ttcgagatgg ttgttggtat attagctgtt180ttaaaagcag gtggtgcgta tttgccaatt gatacagcgt acccgatgca acgaacagaa240tacgtcctgc aaaatagtga ggcaactatt ctcttaacaa aggaatgtta ccttaaggag300tctttagatt ttgaggggga agttttttac ttagatgatg caagactgtt tgaaggggat360agaagagatt tacaaaatat caataatcct actaaccttg cttatatcat ttatacatca420ggatccacgg gaaatccaaa aggtgttatg gtagcgcatc aaagtgttgt gaatttgcta480
ctcgatttac aagagaaata tccggtgcta gcagaagata agcacttgtt aaaaacaaca540tatacgtttg atgtttctgt agccgaaatt tttggatggt ttcatgcagg tggcacactt600gttattgctg gacatggtga tgaaaaagac ccagagaaac tgattcaatt gattcaatgc660cacaaggtta cacatattaa cttcgtacca tcgatgctac atgcaatgtt acaggccttg720gatgaaaaag attttgcaat tatgaatcgg ttgaaatata ttatcgtcgc aggagaagct780gtttcaccag aactttgtaa tcgactgtac gctcattgtc caaatgtaaa actagaaaat840ctatatgggc caacggaagg aacgatttat gcgacagggt tttctattca taaagaaatg900aatgtagcta atgtaccgat tggaaaacca ctttctcatg tggaaacgta tattcttgat960caaaacaatc aaattgtacc aattggtgta ccaggtgaat tgtgtctggg aggaatatgt1020gtagcaaaag gttatatgaa agagccggtg ttaacagaag aaaaattcgt cgtcaatcct1080atgaaacaaa gtgaaagaat gtaccgaacg ggtgatttgg tacgctggtt agcagatggg1140aatattgaat atttaggaag aatagataac caagtcaaga taagaggctt ccgaattgag1200cttggtgaaa ttgaagcggc aattgctgca ttagaagatg tagtacaaac aattgttaca1260acaatgacgg atcataaagg tgcgaacaag attgtcgcat atgttgtgag cgaaaagtat1320gatgaagaac gaattcgtga acatgtgaaa aagacgttgc cgcaatatat ggtaccaagt1380tatttcgttt cgatgaaggc attgcctctt aataaaaatg gaaaagttga tcgcaaacag1440ttgcattcgg ttgatcttta tgaaacgagt atggatacag tcattgtggg accaagaaac1500gaaaaagaag caatgctttc ggttatttgg caagagcttt tgggattaga gaatatcagt1560gttcacgata atttctttaa gcttggtggt cattccatta atgcgacaca attggtatca1620aaaatttata gtgtttgccg agtgagaatg cctcttaaaa atgtgtttca gtatacaacg1680ttagctacaa tggcacgggt gttagaagag ttgttggtaa gcgctgttga cgaagtagct1740gtaacaacgg agcgcattcc aaagatacta ccgagaacat attacgattt gtcgtattca1800
caacaaagaa tttatttctt atctacaatg gagaaagaaa ccaattacta taatattctt1860ggtgcttggg atatttatgg gaaactagat gttacgctat ttgaaaaagc aatccaacta1920ttaatgaaga aacaccattc attacgtgca acatttgaaa tcgtggacgg caaacctgtg1980caaatcatcc acgatgatat ggaaattcct gtgcaattta ttgaccttac tgtgatgcca2040gaaggattac ggatagaaga agtagatgaa cttatgttaa aagagtctaa aagagtatac2100aatctcgcaa atggtccgtt aatgcattgt acaattgtta agataaaaga aggtgagcat2160gtattattga ttggacaaca tcatatcatt agtgatggtt ggtcacttgg tatttttgta2220aaagagttaa atgaaatgta tgatgccttt gtgcaacaca aaccagttgc tgaaacacca2280tcaacaatct ccattatgga ctttactgct tggcacaata gtaaagtaga tgaagatcaa2340gatgatcgac aatattggtt acagcgattt gagggagagt taccgacgtt agagttgccg2400acagacagac aacgtccact tttgaaaaca tatcatggtg acacattatc atataaggtg2460aattctcaat tgcatcaaaa attaaaggac tttagtcatg caaatggtgt aacgatgttt2520atgacgctat taacggcgta taatattatg ttgaataagt taacaaatga aacagacatt2580gttgttggct cccctgtagc aggtagaaat gaaccagaat caaaagattt aatcgggatg2640tttgtgaata cgttagcgtt acgttcgcat ttaggagata atccgacagt tgatgtctta2700ttaaaacaaa taaaacagaa tactttagaa gcatacaatc atcaagatta tccatttgat2760aagttggttg atgacttgga tccacatcga gatttaagta ggacaccaat tttccaagtg2820atgatgggat atatgaatat gccattgatg gttgcatttc gtgaagcaga ggttcgcgaa2880cgatttgttc gacataaagt cgcaaggttt gatttaacac ttcatgtgtt tgaagatgaa2940gatcagatga aaatattctt tgagtataat acagatttat ttgatgaatc aacgattatg3000cgttggcaga atcatttcga aacgctatta caggaaattg tatcgaatcc gacaaaacgt3060atttcggaat tgaatatact tacaaatgag gagaaatatg aaattctaga gatgaacaat3120
aattcaacgg agtatcctca gcatgaatct gttgcggaga tttttagaga aacgaagata3180aagcatcaag caaaactagc aattacgtac aaagatagaa agttaacgta tgcagagttg3240agtgaaaaag caaatgcgtt ggcacataca ttgaaacgtc gaggtgttgc gcagcatgat3300gttgttggaa ttgtcgcaga gcgttcgcct gaaacaatta ttggaatact cgcaatctta3360aaagtaggag caatttattt gccaattgat ccaaaactac cgcaattaac actgcaacac3420atttggcgag atagcggtgc aaaagtcctc ctagggaaaa atgaaacaac tgtagaagtt3480ggcaaggaag ttccgtttgt ggacatcgaa ggggataaag ggaagcaaga ggagttagtg3540tgtccaatta gtccagaaga tacggcatat attatgtata cgtcaggcag tactggaaaa3600ccaaaagggg ttatggtgac acatagaaat attgttcgtt tagtaaaaaa tacgaatttc3660gtttctttgc aagagcaaga tgtgttgtta cagacaggtt cgcttacttt tgacgctgca3720acatttgaaa tttggggcgc attgctaaat ggacttacgc ttcatttagt agaagattac3780gtaattttag atggggaggc gcttcaggaa gagattcagc agaacaaagc aaccattatg3840tgggtgagtg caccgctgtt taatcaattg gcggatcaaa acccagcgat gtttacaggc3900attaagcaat tgctcattgg tggtgatgtt ttatcgccaa aacatattaa caaagtgatg3960gaccattgtg caccaatcaa tatcattaat ggatacggtc caacagaaaa tacgacgttc4020tcgacgtcat ttgtaattga tcaaatgtat caagacagca ttccgattgg aacaccgatt4080gctaattcta gtgcttacat tttagatgta catcaaaata tacaacctat tggtgtagtt4140ggcgaactat gtgttggtgg tgatggagtt gcaaaaggtt atgtgaacct tgaacaatta4200acagaagaac ggtttatagc agatccgttc ctaaagggtt ctacaatgta cagaaccggc4260gattatgtga aattattgcc taatggaaat attcaataca ttggacgtgt ggacaatcaa4320gtgaaaattc gtggattccg catcgaatta gaagccatta tgaacacatt aaaacaatgt4380gaatcaatca aagatgtaat tgttgttgta caagaacaga atgggtataa aacactggtt4440
gcatatgttg tgggagaaga atcgctttca atagaaacag tgagggccta tgcaaaaaaa4500catttggctg aatatatggt accttctcaa tttatattta tagaagaaat tccgctctca4560ataaatggga aagtacagta tagtaagtta ccgaaagtac aagaagtatt gcataaaaaa4620gtagaaacgc tgttaccaga aaacagatta gaagaaatta ttctacgtgt gtatcgtgat4680gtattagaga aagaagattt tggcgtaaca gattcattct tcgcttatgg tggtgactct4740ttactaagta ttcaagtcgt ttcgatgttg aaaaaagagg agattgcagt agatccgaaa4800atgattttta tgcatacaac ggttagagag ttagcaaagg catgtgaaaa tcgtccggtt4860atggaagaaa caaaaaggac tgagaaggat tatttaattc aaatgcgtga aggtagtgaa4920gaagatagtt gtatcatttt tgctcctccg gcaggtggaa cggtacttgg atatatagaa4980ttagcaaggt atttcgaggg aattggcaat gtttacggcc tacaagcacc gggactgtat5040gacgatgaag agcctacgtt cttagattac gatgaacttg tacaagtgtt tcttcgctcg5100attgaaggga catatcgtcc aggtcaagat tatttaggtg gccactcctt agggggacat5160atcgcatttg gaatgtgctg tgaactgatt aagcaaggaa aggcaccaaa gggattgcta5220attctagata caacaccatc acttcaagtt gtaaaggggg ccaaggatga aaaaatagcc5280gaggaggact ttaaaatgat ggtactggct gccggtatcg gaaatatgat gggtgttgat5340ccagaagaat taaagcaact gtcgtatgaa gaagcaaaaa caagagttgt cgcagtggca5400caaaaggatg aaaagttaaa aacttttata aatgaaacat atttggataa gtatttgaag5460ttacaaattc atagtttact aatgtcacga acattagaat tggagaaaac acaattagat5520attccgatta aggtatttaa aacacagttt catacagaag agctagtaga aagatttgat5580gcttggcata actatacaaa tcaagcctgc acattcattg atataccagg cacacatacg5640acgatgatgc gtttaccaca tgtgaaagag gtagcgaaaa aaatagaaga acagctataa5700
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acgaugaugc guuuaccaca ugugaaagag guagcgaaaa aaauagaaga acagcuauaa 57002101021120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40010ggtgaattgt gtctgggagg 202101121120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40011atttttatta agaggcaatg202101221124212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40012gtcaagataa gaggcttccg aatt 242101321120212DNA213人工序列
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223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40013aatggaatga ccaccaagct 202101421120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40014aggaagttcc gtttgtggac 202101521120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40015cacataacct tttgcaactc 202101621120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40016ggcgaactat gtgttggtgg 20
2101721120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40017taaagagtca ccaccataag 202101821120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40018acgtcaggca gtactggaaa 202101921120212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物40019ttcgatgcgg aatccacgaa 202102021143212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物
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221修飾的鹼基222(14)..(14)223I220
221修飾的鹼基222(17)..(17)223I220
221修飾的鹼基222(26)..(26)223I40020ggaattcctt aaaagcagga ggagcatatg tgccgcttga tcc 432102121141212DNA213人工序列220
223人工序列描述用於擴增催吐素合成酶基因特異的DNA片段的PCR引物220
221修飾的鹼基222(12)..(12)223I220
221修飾的鹼基222(15)..(15)223I220
221修飾的鹼基
222(18)..(18)223I220
221修飾的鹼基222(21)..(21)223I220
221修飾的鹼基222(27)..(27)223I220
221修飾的鹼基222(30)..(30)223I220
221修飾的鹼基222(33)..(33)223I40021ggaattcctt taggattacc agttgtacca gaagtgtaaat 418權利要求
1.一種含有序列編號1的胺基酸序列的多肽，或含有序列編號1的胺基酸序列的一部分被改變後構成的序列、而且具有催吐素合成活性的多肽。
2.一種含有序列編號3的胺基酸序列的多肽，或含有序列編號3的胺基酸序列的一部分被改變後構成的序列、而且保持上述多肽所具有的有關催吐素合成的功能構造的多肽。
3.編碼權利要求1所述的任一種多肽的核酸。
4.編碼權利要求2所述的任一種多肽的核酸。
5.保有權利要求3所述的核酸的載體。
6.用權利要求5所述的載體轉化的轉化體。
7.含有序列6的鹼基序列中與催吐素合成活性直接有關的區域的至少部分的序列、或含有與上述區域的鹼基序列相互補的序列的至少部分的核酸。
8.含有序列7鹼基序列的至少部分、或含有與上述鹼基序列互補的序列的至少部分的核酸。
9.含有序列9的鹼基序列中與催吐素合成活性直接有關的區域的鹼基序列互補的序列的至少部分的核酸。
10.含有與序列編號9的鹼基序列互補的序列的至少部分的核酸。
11.被設計成可以對含有在編碼具有催吐素合成活性的多肽的DNA中直接與催吐素合成活性有關的區域的至少部分的DNA區域進行特異擴增的一組核酸。
12.一種將權利要求7所述的核酸固定於不溶性支持體後構成的固相化核酸。
13.一種與催吐素合成酶特異結合的抗體。
14.一種抗體，其特徵是對含有序列編號1的胺基酸序列的多肽具有結合性、而對由序列編號2的胺基酸序列構成的多肽沒有結合性。
15.一種含有權利要求7所述核酸的催吐素檢測用試劑盒。
16.一種含有權利要求11所述的一組核酸、DNA擴增用酶以及DNA合成試劑的催吐素檢測用試劑盒。
17.一種含有權利要求13所述的抗體和抗原抗體反應用試劑的催吐素檢測用試劑盒。
18.一種包括研究檢體中的(a)或(b)存在的步驟的催吐素檢測方法(a)含有序列編號1的胺基酸序列的多肽，或含有序列編號1的胺基酸序列的一部分被改變後構成的序列、而且具有催吐素合成活性的多肽，(b)編碼(a)中任一種多肽的核酸。
19.一種包括以下步驟的催吐素檢測方法(i)以檢體中的DNA為模板，使用權利要求11所述的一組核酸進行DNA擴增反應的步驟，(ii)對被擴增的DNA進行檢測的步驟。
20.一種包括以下步驟的催吐素檢測方法(iii)以檢體中的DNA為模板製備cDNA的步驟，(iv)使用權利要求11所述的一組核酸進行DNA擴增反應的步驟，(v)對被擴增的DNA進行檢測的步驟。
21.一種包括以下步驟的催吐素檢測方法(I)使檢體與權利要求13所述的抗體接觸的步驟，(II)I的步驟後，對抗原抗體反應物進行檢測的步驟。
22.權利要求18所述的催吐素檢測方法，其中作為前處理進行以下步驟(A)將檢體接種到蠟狀芽孢桿菌的增菌培養基中進行培養的步驟。
23.權利要求18所述的催吐素檢測方法，其中作為前處理進行以下步驟(A)將檢體接種到蠟狀芽孢桿菌的增菌培養基中進行培養的步驟，(B)對增殖的蠟狀芽孢桿菌進行溶菌或破碎的步驟。
全文摘要
提供蠟狀芽孢桿菌(Bacillus cereus)產生的嘔吐毒素(催吐素)的簡便而且迅速的檢測法。以檢體中的催吐素合成酶的存在作為指標，對催吐素進行檢測。通過檢測編碼催吐素的核酸、或通過使用對該酶特異的抗體的免疫學方法進行催吐素合成酶的存在的檢測。
文檔編號C07K14/32GK1653173SQ0381126
公開日2005年8月10日 申請日期2003年5月16日 優先權日2002年5月17日
發明者太田美智男, 安形則雄 申請人:股份有限公司生物控制研究所