專利名稱：鉤端螺旋體外膜蛋白及其編碼序列的制作方法
技術(shù)領(lǐng)域：
本發(fā)明屬于生物技術(shù)和醫(yī)學(xué)領(lǐng)域，具體地說(shuō)，本發(fā)明涉及新的編碼鉤端螺旋體外膜蛋白OMP(Outer membrane protein)的多核苷酸，以及此多核苷酸編碼的多肽。本發(fā)明還涉及此多核苷酸和多肽的用途和制備。具體地說(shuō)，本發(fā)明的多肽是一種新的可作為免疫抗原。
背景技術(shù)：
鉤端螺旋體病是一種分布廣泛的人獸共患病，主要分布在中國(guó)南方以及東南亞、南亞等發(fā)展中國(guó)家和地區(qū)。在中國(guó)，本病1995年列入法定傳染病。1955-1993年，年平均發(fā)病率為十萬(wàn)分之七，年平均死亡率占發(fā)病人數(shù)的1.02％。
中國(guó)鉤端螺旋體病的的流行菌群以黃疸出血群和波摩那群為主。黃疸出血群有15個(gè)血清型，其中以賴型最為常見(jiàn)。
中國(guó)雖在該病的防治方面取得了巨大的成功，但對(duì)該病的致病免疫的機(jī)理、疫苗的高效保護(hù)等問(wèn)題仍未解決。
鉤端螺旋體(Leptospira sp.)屬格蘭氏陰性菌，外膜是菌體的一個(gè)組成部分，位于鉤端螺旋體細(xì)胞的最外層，外膜蛋白質(zhì)具有抗原活性，是抗體和補(bǔ)體的主要作用對(duì)象。鉤端螺旋體菌苗是鉤端螺旋體病特異性預(yù)防的一種生物制品，經(jīng)歷了死菌苗、活菌苗、純化菌苗、基因工程重組菌苗的發(fā)展階段。然而，這些疫苗的效果還不令人滿意。
然而目前為止，仍沒(méi)有開(kāi)發(fā)出利用鉤端螺旋體外膜蛋白的免疫制劑。因此，本領(lǐng)域迫切需要開(kāi)發(fā)新的利用鉤端螺旋體外膜蛋白作為抗原的疫苗。

發(fā)明內(nèi)容
本發(fā)明的目的是提供新的鉤端螺旋體外膜蛋白OMP蛋白以及其片段、類似物和衍生物。
本發(fā)明的另一目的是提供編碼這些多肽的多核苷酸。
本發(fā)明的另一目的是提供生產(chǎn)這些多肽的方法以及該多肽和編碼序列的用途。
在本發(fā)明的第一方面，提供新穎的分離出的OMP多肽，它包含具有SEQ IDNO2-26中的偶數(shù)序列和SEQ ID NO27-36氨基酸序列的多肽、或其保守性變異多肽、或其活性片段、或其活性衍生物。較佳地，該多肽是具有SEQ ID NO2-26中的偶數(shù)序列和SEQ ID NO27-36氨基酸序列的多肽，更佳地不含信號(hào)肽序列。
在本發(fā)明的第二方面，提供分離的編碼上述多肽的多核苷酸，該多核苷酸包含一核苷酸序列，該核苷酸序列與選自下組的一種核苷酸序列有至少70％相同性(a)編碼上述鉤端螺旋體OMP多肽的多核苷酸；和(b)與多核苷酸(a)互補(bǔ)的多核苷酸。較佳地，該多核苷酸編碼具有SEQ ID NO2-26中的偶數(shù)序列和SEQ ID NO27-36所示氨基酸序列的多肽。更佳地，該多核苷酸的序列是選自下組的一種(a)具有SEQ ID NO1-26中的奇數(shù)序列。
在本發(fā)明的第三方面，提供了含有上述多核苷酸的載體，以及被該載體轉(zhuǎn)化或轉(zhuǎn)導(dǎo)的宿主細(xì)胞或者被上述多核苷酸直接轉(zhuǎn)化或轉(zhuǎn)導(dǎo)的宿主細(xì)胞。
在本發(fā)明的第四方面，提供了制備鉤端螺旋體OMP蛋白多肽的方法，該方法包含(a)在適合表達(dá)鉤端螺旋體OMP蛋白的條件下，培養(yǎng)上述被轉(zhuǎn)化或轉(zhuǎn)導(dǎo)的宿主細(xì)胞；(b)從培養(yǎng)物中分離出鉤端螺旋體OMP蛋白多肽。
在本發(fā)明的第五方面，提供了與上述的鉤端螺旋體OMP多肽特異性結(jié)合的抗體。還提供了可用于檢測(cè)的核酸分子。
在本發(fā)明的第六方面，提供了檢測(cè)樣品中是否存在OMP蛋白的方法，它包括將樣品與OMP蛋白的特異性抗體接觸，觀察是否形成抗體復(fù)合物，形成了抗體復(fù)合物就表示樣品中存在OMP蛋白。
在本發(fā)明的第七方面，提供了本發(fā)明多肽和編碼序列的用途。例如本發(fā)明的鉤端螺旋體OMP蛋白的編碼序列或其片段，可被作為引物用于PCR擴(kuò)增反應(yīng)，或者作為探針用于雜交反應(yīng)，或者用于制造基因芯片或微陣列。
在本發(fā)明的第八方面，提供了一種疫苗組合物，它含有安全有效量的本發(fā)明的鉤端螺旋體OMP多肽或具片段以及藥學(xué)上可接受的載體。
本發(fā)明的其它方面由于本文的技術(shù)的公開(kāi)，對(duì)本領(lǐng)域的技術(shù)人員而言是顯而易見(jiàn)的。


下列附圖用于說(shuō)明本發(fā)明的具體實(shí)施方案，而不用于限定由權(quán)利要求書(shū)所界定的本發(fā)明范圍。
圖1A-C顯示了本發(fā)明各外膜蛋白的功能結(jié)構(gòu)域。圖中各編號(hào)含義如下PD324953 F1 Immunogenic 7 protein PG4.Porphyromonas gingivalis(Bacteroides gingivalis).
PD000930 F111 OMPA(6)(O83630 Treponema pallidum；YFIB_ECOLI.)
PD005941F13 SERINE PRECURSOR SIGNAL WALL PROTEASE HYDROLASE ENVELOPETRANSMEMBRANE(Q53637_STRAG 581-1150)PD306709F2 OUTER MEMBRANE PROTEOME TPN50(TP50_TREPA，O51189_BORBU)PD77061F1 RPL7A-HSF1 TRANSMEMBRANE(YGH5_YEAST)PD202783F1 GELSOLIN-RELATED(O96923_DICDI 1-1087)PD005701F39 OUTER MEMBRANE PROTEOME，ANTIGEN D15 TP92 SIGNAL PRECURSORSURFACEPD003557F69 COMPLETE PROTEOME OUTER.MEMBRANE ANTIGEN D15 SIGNAL OMP85PRECURSOR SURFACEPD134379F1LIPOPROTEIN LIPL36 MEMBRANEPD124660F2LIPOPROTEIN LIPL32 MEMBRANE(LEPTO LAI)PD41611F2LIPOPROTEIN MEMBRANE OUTERPD323220F1HYPOTHETICAL 45.9kDa PROTEINPD416752F2PROTEOME COMPLETE BINDING PA4035 DOMAIN PEPTIDOGLYCANPD000002F1172MYOSIN CHAIN COIL COILED HEAVY MUSCLE ATP-BINDINGMULTIGENE FAMILY ACTIN-BINDING(HUMAN.MOUSE)PD140481F1 O67180_Aquifex aeolicusPD130335F1 P74182_SynechocystisPD43188 F1 YMM8 Yeast Coxl4-HmgsPD134743F1 O66516_Aquifex aeolicus.
PD000002F1172 MYOSIN CHAIN COIL COILED HEAVY MUSCLE ATP-BINDINGMULTIGENE FAMILY ACTIN-BINDING(HUMAN，MOUSE)PD008899 LRR PD085687 LRR F350PD038310F3OUTER LAYER FLAA(2)BORBU 188-297PD038310F3OUTER LAYER FLAA(2)BORBU 188-29具體實(shí)施方式
本發(fā)明人經(jīng)過(guò)廣泛而深入的研究，從問(wèn)號(hào)鉤端螺旋體(Leptospirainterrogans)黃疸出血群賴型賴株中分離純化和鑒定了一類新的外膜蛋白質(zhì)，該外膜蛋白可作為抗原用于疫苗的設(shè)計(jì)和制備。在此基礎(chǔ)上完成了本發(fā)明。
在本發(fā)明中，術(shù)語(yǔ)“OMP蛋白”、“OMP多肽”或“鉤端螺旋體外膜蛋白OMP”可互換使用，都指具有鉤端螺旋體外膜蛋白OMP氨基酸序列(SEQ ID NO226中的偶數(shù)序列和SEQ ID NO27-36)的蛋白或多肽。它們包括含有或不含起始甲硫氨酸的鉤端螺旋體外膜蛋白OMP，以及含有或不含有信號(hào)肽的外膜蛋白。
如本文所用，“分離的”是指物質(zhì)從其原始環(huán)境中分離出來(lái)(如果是天然的物質(zhì)，原始環(huán)境即是天然環(huán)境)。如活體細(xì)胞內(nèi)的天然狀態(tài)下的多聚核苷酸和多肽是沒(méi)有分離純化的，但同樣的多聚核苷酸或多肽如從天然狀態(tài)中同存在的其他物質(zhì)中分開(kāi)，則為分離純化的。
如本文所用，“分離的OMP蛋白或多肽”是指OMP多肽基本上不含天然與其相關(guān)的其它蛋白、脂類、糖類或其它物質(zhì)。本領(lǐng)域的技術(shù)人員能用標(biāo)準(zhǔn)的蛋白質(zhì)純化技術(shù)純化OMP蛋白。
本發(fā)明的多肽可以是重組多肽、天然多肽、合成多肽，優(yōu)選重組多肽。本發(fā)明的多肽可以是天然純化的產(chǎn)物，或是化學(xué)合成的產(chǎn)物，或使用重組技術(shù)從原核或真核宿主(例如，細(xì)菌、酵母、高等植物、昆蟲(chóng)和哺乳動(dòng)物細(xì)胞)中產(chǎn)生。根據(jù)重組生產(chǎn)方案所用的宿主，本發(fā)明的多肽可以是糖基化的，但優(yōu)選的是非糖基化的。
本發(fā)明還包括鉤端螺旋體OMP蛋白的片段、衍生物和類似物。如本文所用，術(shù)語(yǔ)“片段”、“衍生物”和“類似物”是指基本上保持本發(fā)明的天然鉤端螺旋體OMP蛋白相同的生物學(xué)功能或活性的多肽。本發(fā)明的多肽片段、衍生物或類似物可以是(i)有一個(gè)或多個(gè)保守或非保守性氨基酸殘基(優(yōu)選保守性氨基酸殘基)被取代的多肽，而這樣的取代的氨基酸殘基可以是也可以不是由遺傳密碼編碼的，或(ii)在一個(gè)或多個(gè)氨基酸殘基中具有取代基團(tuán)的多肽，或(iii)成熟多肽與另一個(gè)化合物(比如延長(zhǎng)多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前導(dǎo)序列或分泌序列或用來(lái)純化此多肽的序列或蛋白原序列，或與抗原IgG片段的形成的融合蛋白)。根據(jù)本文的教導(dǎo)，這些片段、衍生物和類似物屬于本領(lǐng)域熟練技術(shù)人員公知的范圍。
在本發(fā)明中，術(shù)語(yǔ)“鉤端螺旋體OMP多肽”指具有鉤端螺旋體OMP蛋白活性或免疫原性的SEQ ID NO2-26中的偶數(shù)序列和SEQ ID NO27-36序列的多肽。該術(shù)語(yǔ)還包括具有與鉤端螺旋體OMP蛋白相同功能的、上述序列的變異形式。這些變異形式包括(但并不限于)若干個(gè)(通常為1-50個(gè)，較佳地1-30個(gè)，更佳地1-20個(gè)，最佳地1-10個(gè))氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一個(gè)或數(shù)個(gè)(通常為10個(gè)以內(nèi)，更佳地為5個(gè)以內(nèi))氨基酸。該術(shù)語(yǔ)還包括鉤端螺旋體OMP蛋白的活性片段和活性衍生物。
該多肽的變異形式包括同源序列、保守性變異體、等位變異體、天然突變體、誘導(dǎo)突變體、在高或低的嚴(yán)緊度條件下能與鉤端螺旋體OMP DNA雜交的DNA所編碼的蛋白、以及利用抗鉤端螺旋體OMP多肽的抗血清獲得的多肽或蛋白。本發(fā)明還提供了其他多肽，如包含鉤端螺旋體OMP多肽或其片段的融合蛋白。除了幾乎全長(zhǎng)的多肽外，本發(fā)明還包括了鉤端螺旋體OMP多肽的可溶性片段。通常，該片段具有鉤端螺旋體OMP多肽序列的至少約10個(gè)連續(xù)氨基酸，通常至少約30個(gè)連續(xù)氨基酸，較佳地至少約50個(gè)連續(xù)氨基酸，更佳地至少約80個(gè)連續(xù)氨基酸，最佳地至少約100個(gè)連續(xù)氨基酸。這些片段也可用作免疫原以誘導(dǎo)針對(duì)鉤端螺旋體的免疫應(yīng)答反應(yīng)。
發(fā)明還提供鉤端螺旋體OMP蛋白或多肽的類似物。這些類似物與天然鉤端螺旋體OMP多肽的差別可以是氨基酸序列上的差異，也可以是不影響序列的修飾形式上的差異，或者兼而有之。這些多肽包括天然或誘導(dǎo)的遺傳變異體。誘導(dǎo)變異體可以通過(guò)各種技術(shù)得到，如通過(guò)輻射或暴露于誘變劑而產(chǎn)生隨機(jī)誘變，還可通過(guò)定點(diǎn)誘變法或其他已知分子生物學(xué)的技術(shù)。類似物還包括具有不同于天然L-氨基酸的殘基(如D-氨基酸)的類似物，以及具有非天然存在的或合成的氨基酸(如B、γ-氨基酸)的類似物。應(yīng)理解，本發(fā)明的多肽并不限于上述例舉的代表性的多肽。
修飾(通常不改變一級(jí)結(jié)構(gòu))形式包括體內(nèi)或體外的多肽的化學(xué)衍生形式如乙?；螋然Ｐ揎椷€包括糖基化，如那些在多肽的合成和加工中或進(jìn)一步加工步驟中進(jìn)行糖基化修飾而產(chǎn)生的多肽。修飾形式還包括具有磷酸化氨基酸殘基(如磷酸酪氨酸，磷酸絲氨酸，磷酸蘇氨酸)的序列。還包括被修飾從而提高了其抗蛋白水解性能或優(yōu)化了溶解性能的多肽。
在本發(fā)明中，“鉤端螺旋體OMP蛋白保守性變異多肽”指與SEQ ID NO2-26中的偶數(shù)序列和SEQ ID NO27-36的氨基酸序列相比，有至多10個(gè)，較佳地至多8個(gè)，更佳地至多5個(gè)，最佳地至多3個(gè)氨基酸被性質(zhì)相似或相近的氨基酸所替換而形成多肽。這些保守性變異多肽最好根據(jù)表1進(jìn)行氨基酸替換而產(chǎn)生。
表1


本發(fā)明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因組DNA或人工合成的DNA。DNA可以是單鏈的或是雙鏈的。DNA可以是編碼鏈或非編碼鏈。以外膜蛋白LA56為例，編碼成熟多肽的編碼區(qū)序列可以與SEQ ID NO1所示的編碼區(qū)序列相同或者是簡(jiǎn)并的變異體。如本文所用，以外膜蛋白LA56為例，“簡(jiǎn)并的變異體”在本發(fā)明中是指編碼具有SEQ ID NO2的蛋白質(zhì)，但與SEQ ID NO1所示的編碼區(qū)序列有差別的核酸序列。本發(fā)明的其他蛋白依此類推。本發(fā)明的核苷酸序列可以根據(jù)密碼子的對(duì)應(yīng)關(guān)系，根據(jù)SEQ ID NO2-26中的偶數(shù)序列和SEQ IDNO27-36的氨基酸推導(dǎo)出。
編碼成熟多肽的多核苷酸包括只編碼成熟多肽的編碼序列；成熟多肽的編碼序列和各種附加編碼序列；成熟多肽的編碼序列(和任選的附加編碼序列)以及非編碼序列。
術(shù)語(yǔ)“編碼多肽的多核苷酸”可以是包括編碼此多肽的多核苷酸，也可以是還包括附加編碼和/或非編碼序列的多核苷酸。
本發(fā)明還涉及上述多核苷酸的變異體，其編碼與本發(fā)明有相同的氨基酸序列的多肽或多肽的片段、類似物和衍生物。此多核苷酸的變異體可以是天然發(fā)生的等位變異體或非天然發(fā)生的變異體。這些核苷酸變異體包括取代變異體、缺失變異體和插入變異體。如本領(lǐng)域所知的，等位變異體是一個(gè)多核苷酸的替換形式，它可能是一個(gè)或多個(gè)核苷酸的取代、缺失或插入，但不會(huì)從實(shí)質(zhì)上改變其編碼的多肽的功能。
本發(fā)明還涉及與上述的序列雜交且兩個(gè)序列之間具有至少50％，較佳地至少70％，更佳地至少80％相同性的多核苷酸。本發(fā)明特別涉及在嚴(yán)格條件下與本發(fā)明所述多核苷酸可雜交的多核苷酸。在本發(fā)明中，“嚴(yán)格條件”是指(1)在較低離子強(qiáng)度和較高溫度下的雜交和洗脫，如0.2×SSC，0.1％SDS，60℃；或(2)雜交時(shí)加有變性劑，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)僅在兩條序列之間的相同性至少在90％以上，更好是95％以上時(shí)才發(fā)生雜交。并且，可雜交的多核苷酸編碼的多肽與成熟的鉤端螺旋體外膜蛋白有相同的生物學(xué)功能和活性。
本發(fā)明還涉及與上述的序列雜交的核酸片段。如本文所用，“核酸片段”的長(zhǎng)度至少含15個(gè)核苷酸，較好是至少30個(gè)核苷酸，更好是至少50個(gè)核苷酸，最好是至少100個(gè)核苷酸以上。核酸片段可用于核酸的擴(kuò)增技術(shù)(如PCR)以確定和/或分離編碼OMP蛋白的多聚核苷酸。
本發(fā)明的鉤端螺旋體OMP核苷酸全長(zhǎng)序列或其片段通?？梢杂肞CR擴(kuò)增法、重組法或人工合成的方法獲得。對(duì)于PCR擴(kuò)增法，可根據(jù)本發(fā)明所公開(kāi)的有關(guān)核苷酸序列，尤其是開(kāi)放閱讀框序列來(lái)設(shè)計(jì)引物，并用市售的DNA文庫(kù)或按本領(lǐng)域技術(shù)人員已知的常規(guī)方法所制備的DNA文庫(kù)作為模板，擴(kuò)增而得有關(guān)序列。當(dāng)序列較長(zhǎng)時(shí)，常常需要進(jìn)行兩次或多次PCR擴(kuò)增，然后再將各次擴(kuò)增出的片段按正確次序拼接在一起。
一旦獲得了有關(guān)的序列，就可以用重組法來(lái)大批量地獲得有關(guān)序列。這通常是將其克隆入載體，再轉(zhuǎn)入細(xì)胞，然后通過(guò)常規(guī)方法從增殖后的宿主細(xì)胞中分離得到有關(guān)序列。
此外，還可用人工合成的方法來(lái)合成有關(guān)序列，尤其是片段長(zhǎng)度較短時(shí)。通常，通過(guò)先合成多個(gè)小片段，然后再進(jìn)行連接可獲得序列很長(zhǎng)的片段。
目前，已經(jīng)可以完全通過(guò)化學(xué)合成來(lái)得到編碼本發(fā)明蛋白(或其片段，或其衍生物)的DNA序列。然后可將該DNA序列引入本領(lǐng)域中已知的各種現(xiàn)有的DNA分子(或如載體)和細(xì)胞中。此外，還可通過(guò)化學(xué)合成將突變引入本發(fā)明蛋白序列中。
本發(fā)明也涉及包含本發(fā)明的多核苷酸的載體，以及用本發(fā)明的載體或OMP蛋白編碼序列經(jīng)基因工程產(chǎn)生的宿主細(xì)胞，以及經(jīng)重組技術(shù)產(chǎn)生本發(fā)明所述多肽的方法。
通過(guò)常規(guī)的重組DNA技術(shù)(Science，1984；2241431)，可利用本發(fā)明的多聚核苷酸序列可用來(lái)表達(dá)或生產(chǎn)重組的OMP多肽。一般來(lái)說(shuō)有以下步驟(1).用本發(fā)明的編碼鉤端螺旋體OMP多肽的多核苷酸(或變異體)，或用含有該多核苷酸的重組表達(dá)載體轉(zhuǎn)化或轉(zhuǎn)導(dǎo)合適的宿主細(xì)胞；(2).在合適的培養(yǎng)基中培養(yǎng)的宿主細(xì)胞；(3).從培養(yǎng)基或細(xì)胞中分離、純化蛋白質(zhì)。
本發(fā)明中，鉤端螺旋體OMP多核苷酸序列可插入到重組表達(dá)載體中。術(shù)語(yǔ)“重組表達(dá)載體”指本領(lǐng)域熟知的細(xì)菌質(zhì)粒、噬菌體、酵母質(zhì)粒、植物細(xì)胞病毒、哺乳動(dòng)物細(xì)胞病毒如腺病毒、逆轉(zhuǎn)錄病毒或其他載體。在本發(fā)明中適用的載體包括但不限于在細(xì)菌中表達(dá)的基于T7的表達(dá)載體；在哺乳動(dòng)物細(xì)胞中表達(dá)的pMSXND表達(dá)載體和在昆蟲(chóng)細(xì)胞中表達(dá)的來(lái)源于桿狀病毒的載體?？傊灰茉谒拗黧w內(nèi)復(fù)制和穩(wěn)定，任何質(zhì)粒和載體都可以用。表達(dá)載體的一個(gè)重要特征是通常含有復(fù)制起點(diǎn)、啟動(dòng)子、標(biāo)記基因和翻譯控制元件。
本領(lǐng)域的技術(shù)人員熟知的方法能用于構(gòu)建含鉤端螺旋體OMP編碼DNA序列和合適的轉(zhuǎn)錄/翻譯控制信號(hào)的表達(dá)載體。這些方法包括體外重組DNA技術(shù)、DNA合成技術(shù)、體內(nèi)重組技術(shù)等。所述的DNA序列可有效連接到表達(dá)載體中的適當(dāng)啟動(dòng)子上，以指導(dǎo)mRNA合成。表達(dá)載體還包括翻譯起始用的核糖體結(jié)合位點(diǎn)和轉(zhuǎn)錄終止子。
此外，表達(dá)載體優(yōu)選地包含一個(gè)或多個(gè)選擇性標(biāo)記基因，以提供用于選擇轉(zhuǎn)化的宿主細(xì)胞的表型性狀，如真核細(xì)胞培養(yǎng)用的二氫葉酸還原酶、新霉素抗性以及綠色熒光蛋白(GFP)，或用于大腸桿菌的四環(huán)素或氨芐青霉素抗性。
包含上述的適當(dāng)DNA序列以及適當(dāng)啟動(dòng)子或者控制序列的載體，可以用于轉(zhuǎn)化適當(dāng)?shù)乃拗骷?xì)胞，以使其能夠表達(dá)蛋白質(zhì)。
宿主細(xì)胞可以是原核細(xì)胞，如細(xì)菌細(xì)胞；或是低等真核細(xì)胞，如酵母細(xì)胞；或是高等真核細(xì)胞，如哺乳動(dòng)物細(xì)胞。代表性例子有大腸桿菌，鏈霉菌屬；鼠傷寒沙門氏菌的細(xì)菌細(xì)胞；真菌細(xì)胞如酵母；植物細(xì)胞；果蠅S2或Sf9的昆蟲(chóng)細(xì)胞；CHO的動(dòng)物細(xì)胞等。
用重組DNA轉(zhuǎn)化宿主細(xì)胞可用本領(lǐng)域技術(shù)人員熟知的常規(guī)技術(shù)進(jìn)行。當(dāng)宿主為原核生物如大腸桿菌時(shí)，能吸收DNA的感受態(tài)細(xì)胞可在指數(shù)生長(zhǎng)期后收獲，用CaCl2法處理，所用的步驟在本領(lǐng)域眾所周知。另一種方法是使用MgCl2。如果需要，轉(zhuǎn)化也可用電穿孔的方法進(jìn)行。當(dāng)宿主是真核生物，可選用如下的DNA轉(zhuǎn)染方法磷酸鈣共沉淀法，常規(guī)機(jī)械方法如顯微注射、電穿孔、脂質(zhì)體包裝等。
獲得的轉(zhuǎn)化子可以用常規(guī)方法培養(yǎng)，表達(dá)本發(fā)明的基因所編碼的多肽。根據(jù)所用的宿主細(xì)胞，培養(yǎng)中所用的培養(yǎng)基可選自各種常規(guī)培養(yǎng)基。在適于宿主細(xì)胞生長(zhǎng)的條件下進(jìn)行培養(yǎng)。當(dāng)宿主細(xì)胞生長(zhǎng)到適當(dāng)?shù)募?xì)胞密度后，用合適的方法(如溫度轉(zhuǎn)換或化學(xué)誘導(dǎo))誘導(dǎo)選擇的啟動(dòng)子，將細(xì)胞再培養(yǎng)一段時(shí)間。
在上面的方法中的重組多肽可在細(xì)胞內(nèi)、或在細(xì)胞膜上表達(dá)、或分泌到細(xì)胞外。如果需要，可利用其物理的、化學(xué)的和其它特性通過(guò)各種分離方法分離和純化重組的蛋白。這些方法是本領(lǐng)域技術(shù)人員所熟知的。這些方法的例子包括但并不限于常規(guī)的復(fù)性處理、用蛋白沉淀劑處理(鹽析方法)、離心、滲透破菌、超處理、超離心、分子篩層析(凝膠過(guò)濾)、吸附層析、離子交換層析、高效液相層析(HPLC)和其它各種液相層析技術(shù)及這些方法的結(jié)合。
重組的鉤端螺旋體OMP蛋白或多肽有多方面的用途，其中包括(但不限于)作為抗原用于制備疫苗。
另一方面，本發(fā)明還包括對(duì)鉤端螺旋體OMP DNA或是其片段編碼的多肽具有特異性的多克隆抗體和單克隆抗體，尤其是單克隆抗體。這里，“特異性”是指抗體能結(jié)合于鉤端螺旋體OMP基因產(chǎn)物或片段。較佳地，指那些能與鉤端螺旋體OMP基因產(chǎn)物或片段結(jié)合但不識(shí)別和結(jié)合于其它非相關(guān)抗原分子的抗體。本發(fā)明還包括那些能與修飾或未經(jīng)修飾形式的鉤端螺旋體OMP基因產(chǎn)物結(jié)合的抗體。
本發(fā)明不僅包括完整的單克隆或多克隆抗體，而且還包括具有免疫活性的抗體片段，如Fab′或(Fab)2片段；抗體重鏈；抗體輕鏈；或嵌合抗體。
本發(fā)明的抗體可以通過(guò)本領(lǐng)域內(nèi)技術(shù)人員已知的各種技術(shù)進(jìn)行制備。例如，純化的鉤端螺旋體OMP基因產(chǎn)物或者其具有抗原性的片段，可被施用于動(dòng)物以誘導(dǎo)多克隆抗體的產(chǎn)生。與之相似的，表達(dá)鉤端螺旋體OMP蛋白或其具有抗原性的片段的細(xì)胞可用來(lái)免疫動(dòng)物來(lái)生產(chǎn)抗體。本發(fā)明的抗體也可以是單克隆抗體。此類單克隆抗體可以利用雜交瘤技術(shù)來(lái)制備。
疫苗組合物本發(fā)明的疫苗可以是預(yù)防性的(即預(yù)防感染)或治療性的(即在感染后治療疾病)。
這些疫苗包含免疫性抗原或免疫原、免疫原性多肽、蛋白或蛋白片段、或核酸(如核糖核酸或脫氧核糖核酸)，通常與″藥學(xué)上可接受的載體″組合，這些載體包括本身不誘導(dǎo)產(chǎn)生對(duì)接受該組合物的個(gè)體有害的抗體的任何載體。合適的載體通常是大的、代謝緩慢的大分子，如蛋白質(zhì)、多糖、聚乳酸、聚乙醇酸、氨基酸聚合物、氨基酸共聚物、脂質(zhì)凝集物(如油滴或脂質(zhì)體)以及無(wú)活性的病毒顆粒。這些載體是本領(lǐng)域普通技術(shù)人員所熟知的。另外，這些載體可起免疫刺激劑(″佐劑″)作用。另外，抗原或免疫原可以和細(xì)菌類毒素(如白喉、破傷風(fēng)、霍亂、幽門螺桿菌等病原體的類毒素)偶聯(lián)。
增強(qiáng)組合物效果的較佳的佐劑包括但不局限于(1)鋁鹽(alum)，如氫氧化鋁、磷酸鋁、硫酸鋁等；(2)水包油型乳劑配方(有或沒(méi)有其它特異性的免疫刺激劑，如胞壁酰肽(見(jiàn)下文)或細(xì)菌細(xì)胞壁成分)，例如，(a)MF59(PCT出版物No.WO90/14837)，其含有5％鯊烯、0.5％吐溫80和0.5％Span 85(任選地含有不同量的MTP-PE(見(jiàn)下文)，雖然并不需要)，用微量流化器(如110Y型微量流化器(Microfluidics，Newton，MA))制成亞微米級(jí)顆粒；(b)SAF，其含有10％鯊烯、0.4％吐溫80、5％普盧蘭尼克(pluronic)嵌段聚合物L(fēng)121以及thr-MDP(見(jiàn)下文)，微量流化成亞微米級(jí)乳劑或渦流振蕩產(chǎn)生粒徑較大的乳劑，和(c)RibiTM佐劑系統(tǒng)(RAS)(Ribi Immunochem，Hamilton，MT)，其含有2％鯊烯、0.2％吐溫80以及取自單磷酰脂A(MPL)、二霉菌酸海藻糖酯(TDM)、和細(xì)胞壁骨架(CWS)的一種或多種細(xì)菌細(xì)胞壁組分，較佳的是MPL+CWS(DetoxTM)；(3)皂素佐劑，例如可采用StimulonTM(Cambridge Bioscience，Worcester，MA)或從其產(chǎn)生的顆粒，如ISCOM(免疫刺激性復(fù)合物)；(4)Freund完全佐劑(CFA)和Freund不完全佐劑(IFA)；(5)細(xì)胞因子，如白介素(如IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL12等)、干擾素(如γ干擾素)、巨噬細(xì)胞集落刺激因子(M-CFS)、腫瘤壞死因子(TNF)等；(6)細(xì)菌ADP-核糖基化毒素(如霍亂毒素CT，百日咳毒素PT或大腸桿菌熱不穩(wěn)定毒素LT)的脫毒變異體，尤其是LT-K63、LT-R72，CT-S109、PT-K9/G129；參見(jiàn)例如WO93/13302和WO92/19265；以及(7)作為免疫刺激劑來(lái)增強(qiáng)組合物效果的其它物質(zhì)。
如上所述，胞壁酰肽包括但不局限于，N-乙酰-胞壁酰-L-蘇氨酰-D-異谷氨酰胺(thr-MDP)、N-乙酰-去胞壁酰-L-丙氨酰-D-異谷氨酰胺(nor-MDP)、N-乙酰胞壁酰-L-丙氨酰-D-異谷氨酰氨?；?L-丙氨酸-2-(1′-2′-二棕櫚酰-sn-甘油-3-羥基磷酰氧)-乙胺(MTP-PE)等。
包括免疫原性組合物在內(nèi)的疫苗組合物(如可包括抗原，藥學(xué)上可接受的載體以及佐劑)，通常含有稀釋劑，如水，鹽水，甘油，乙醇等。另外，輔助性物質(zhì)，如潤(rùn)濕劑或乳化劑、pH緩沖物質(zhì)等可存在于這類運(yùn)載體中。此外，包括免疫原性組合物在內(nèi)的疫苗組合物可含有在藥學(xué)上可接受載體中的抗原、多肽、蛋白、蛋白片段或核酸。
更具體地，包括免疫原性組合物在內(nèi)的疫苗，包含免疫學(xué)有效量的免疫原性多肽，以及上述其它所需的組分?！迕庖邔W(xué)有效量″指以單劑或連續(xù)劑一部分給予個(gè)體的量對(duì)治療或預(yù)防是有效的。該用量根據(jù)所治療個(gè)體的健康狀況和生理狀況、所治療個(gè)體的類別(如非人靈長(zhǎng)類等)、個(gè)體免疫系統(tǒng)合成抗體的能力、所需的保護(hù)程度、疫苗的配制、治療醫(yī)師對(duì)醫(yī)療狀況的評(píng)估、及其它的相關(guān)因素而定。預(yù)計(jì)該用量將在相對(duì)較寬的范圍內(nèi)，可通過(guò)常規(guī)實(shí)驗(yàn)來(lái)確定。
通常，可將疫苗組合物或免疫原性組合物制成可注射劑，例如液體溶液或懸液；還可制成在注射前適合配入溶液或懸液、液體賦形劑的固體形式。該制劑還可乳化或包封在脂質(zhì)體中，在上述藥學(xué)上可接受的載體下增強(qiáng)佐劑效果。
常規(guī)方法是從腸胃外(皮下或肌內(nèi))途徑通過(guò)注射給予免疫原性組合物。適合其它給藥方式的其它配方包括口服制劑。治療劑量可以是單劑方案或多劑方案。疫苗可以結(jié)合其它免疫調(diào)節(jié)劑一起給予。
作為以蛋白質(zhì)為基礎(chǔ)的疫苗的一種替代方案是，還可以采用DNA疫苗接種。
下面結(jié)合具體實(shí)施例，進(jìn)一步闡述本發(fā)明。應(yīng)理解，這些實(shí)施例僅用于說(shuō)明本發(fā)明而不用于限制本發(fā)明的范圍。下列實(shí)施例中未注明具體條件的實(shí)驗(yàn)方法，通常按照常規(guī)條件如Sambrook等人，分子克隆實(shí)驗(yàn)室手冊(cè)(New YorkColdSpring Harbor Laboratory Press，1989)中所述的條件，或按照制造廠商所建議的條件。
實(shí)施例1菌體培養(yǎng)1)培養(yǎng)基使用EMJH液體培養(yǎng)基，配制5×的EMJH培養(yǎng)基1000毫升，稱取BSA50g。無(wú)水醋酸鈉0.5g，丙酮酸鈉1g，硫酸亞鐵0.25g，1.5％的氯化鎂3.5ml，0.4％的硫酸鋅5ml，0.15的硫酸銅1ml，磷酸氫二鈉12.6g，磷酸二氫鉀1.5g，氯化鈉5g，氯化銨1.25g，0.05％的VB122ml，VB10.025g，調(diào)pH為7.2-7.6，定容至1000ml分別以0.7、0.45、0.2nm孔徑的硝酸纖維素膜過(guò)濾除菌。使用時(shí)，用無(wú)菌水其5倍稀釋。
2)培養(yǎng)條件30℃，100轉(zhuǎn)/分鐘搖動(dòng)培養(yǎng)40小時(shí)。
3)菌株問(wèn)號(hào)鉤端螺旋體黃疸出血群賴型賴株。
實(shí)施例2外膜蛋白質(zhì)提取按以下步驟提取外膜蛋白質(zhì)1)約100mg濕重活菌懸浮在0.5ml 50mM Tris-HCL(pH7.3)中。
2)在French Press壓力細(xì)胞破碎儀中，采用20000psi破碎細(xì)胞三次，至溶液澄清。
3)2500g離心10分鐘，取上清。
4)上清加入4.5ml碳酸鈉(pH11)稀釋，搖勻，置于冰上1小時(shí)。
5)11500g離心1小時(shí)。
6)取沉淀，用1ml Tris-HCL(pH7.3)洗一次。
7)11500g離心20分鐘。
8)沉淀中即包含外膜蛋白質(zhì)(混合物)。
對(duì)外膜蛋白質(zhì)的混合物進(jìn)行SDS-PAGE電泳，條件是4％濃縮膠，12.5％分離膠，從而分離出各外膜蛋白。
實(shí)施例3膠內(nèi)蛋白質(zhì)酶解電泳膠中各條帶分別切下，置于50％水-40％甲醇-10％醋酸中漂洗脫色后，凍干。依次加入5mM二硫蘇糖醇用水泡漲后，搗碎置于100mM碳酸氫氨(pH8.3)，加入TPCK-胰蛋白酶，37℃保溫24小時(shí)。酶解后，凝膠內(nèi)蛋白質(zhì)經(jīng)0.1％三氟乙酸-60％ACN(乙睛)抽提2-3次后，凍干濃縮。
實(shí)施例4液相色譜-電噴霧離子阱質(zhì)譜鑒定蛋白質(zhì)和數(shù)據(jù)庫(kù)檢索測(cè)定在Finnigan MAT LCQ離子阱質(zhì)譜儀上進(jìn)行，毛細(xì)管溫度，200℃；噴霧電壓，4.25kV；鞘氣流速，50；聯(lián)用的HPLC為HP-1100型，反相C8柱(RP-300，ABI公司產(chǎn)品)，50×1.0I.D.mm，流速0.05ml/min；洗脫液，A液，0.01％TFA-1％HAc-H2O；B液，0.01％TFA-1％HAc-ACN。MS/MS能量為42％-45％。
經(jīng)質(zhì)譜鑒定和分析，采用質(zhì)譜測(cè)序功能，獲得了各外膜蛋白的片段的氨基酸序列。隨后根據(jù)獲得的段肽的氨基酸序列，采用Sequest軟件，在本申清人構(gòu)建的蛋白質(zhì)數(shù)據(jù)庫(kù)中檢索，得到唯一的開(kāi)放閱讀框的序列。再根據(jù)開(kāi)放閱讀框的序列，得到編碼蛋白的氨基酸序列。
實(shí)施例5外膜蛋白的核酸序列和氨基酸序列鑒于某些外膜蛋白的表達(dá)豐度較低，難以直接通過(guò)蛋白組學(xué)方法獲得氨基酸序列。因此，在本實(shí)施例中，還用常規(guī)方法抽提鉤端螺旋體DNA，構(gòu)建文庫(kù)。測(cè)定基因組全序列。根據(jù)計(jì)算機(jī)分析結(jié)果，合成引物，以抽提的鉤端螺旋體DNA為模板，進(jìn)行PCR擴(kuò)增，獲得外膜蛋白的ORF序列。再次測(cè)序驗(yàn)證，與基因組序列相符。
通過(guò)實(shí)施例1-4和5共獲得23個(gè)外膜蛋白及其編碼序列，它們編碼具有SEQID NO2-26中的偶數(shù)序列和SEQ ID NO27-36所示氨基酸序列的多肽。本發(fā)明的23種外膜蛋白的基本情況如下表所示。
表A外膜蛋白的核酸序列和氨基酸序列


注(1)帶“*”者的蛋白是通過(guò)實(shí)施例1-4獲得的氨基酸序列。
(2)信號(hào)肽一欄中給出的數(shù)字是信號(hào)肽的起始和終止氨基酸位置。
其中LA3366，LA3367為鉤端螺旋體以及螺旋體所特有的外層鞭毛蛋白，LA3601含有螺旋體外膜蛋白特有的結(jié)構(gòu)域。外膜蛋白No.1-23與其他細(xì)菌的外膜蛋白質(zhì)都有一定的同源性(圖1)，證實(shí)它們是外膜蛋白，其中LA2311(OMP1)，LA490(LipL36)與許多致病性的微生物OMP1和LipL36相似。本發(fā)明的外膜蛋白可用于開(kāi)發(fā)高效疫苗。
實(shí)施例6蛋白表達(dá)用常規(guī)基因工程方法將鉤端螺旋體外膜蛋白基因克隆入融合表達(dá)載體pET28b(Novagene公司)，融合表達(dá)的受體菌選用大腸桿菌BL21(DE3)。常規(guī)條件發(fā)酵后，提取重組菌體蛋白，然后進(jìn)行SDS-PAGE電泳，分子量與預(yù)測(cè)值基本相同。大腸桿菌中表達(dá)的外膜蛋白(帶有His-tag的融合蛋白)，通過(guò)Ni-NTA柱分離純化，得到純的鉤端螺旋體外膜蛋白(融合蛋白)。
實(shí)施例7制備抗體將實(shí)施例6中獲得的純化的、重組His-融合蛋白用來(lái)免疫動(dòng)物以產(chǎn)生抗體，具體方法如下將融合蛋白用等量的完全Freund′s佐劑乳化。用100μg乳化過(guò)的蛋白，對(duì)幼齡豚鼠(體重小于200克)進(jìn)行淋巴結(jié)注射。二周后，用等量的非完全Freund′s佐劑乳化的同樣抗原，對(duì)幼齡豚鼠以100μg的劑量進(jìn)行淋巴結(jié)注射以加強(qiáng)免疫。每隔10天進(jìn)行一次加強(qiáng)免疫，至少進(jìn)行三次。然后用鉤端螺旋體有毒株進(jìn)行感染，結(jié)果豚鼠的感染情況。發(fā)現(xiàn)本發(fā)明蛋白可有效地引起抗鉤端螺旋體的免疫應(yīng)答，從而保護(hù)豚鼠免受鉤端螺旋體感染。
此外，從免疫豚鼠獲得的抗血清的特異反應(yīng)活性用ELISA檢驗(yàn)抗體滴度。結(jié)果發(fā)現(xiàn)，抗體可特異性地與本發(fā)明蛋白發(fā)生結(jié)合。
實(shí)施例8免疫制劑用常規(guī)基因工程方法將膜蛋白基因克隆入融合表達(dá)載體，融合表達(dá)的受體菌選用大腸桿菌或結(jié)核桿菌(卡介苗)。在大腸桿菌中表達(dá)的外膜蛋白須先將其分離、濃縮、提純，加入福氏佐劑制備成膜蛋白免疫原。在卡介苗中表達(dá)的膜蛋白，可直接作為免疫原，即疫苗。
在本發(fā)明提及的所有文獻(xiàn)都在本申請(qǐng)中引用作為參考，就如同每一篇文獻(xiàn)被單獨(dú)引用作為參考那樣。此外應(yīng)理解，在閱讀了本發(fā)明的上述講授內(nèi)容之后，本領(lǐng)域技術(shù)人員可以對(duì)本發(fā)明作各種改動(dòng)或修改，這些等價(jià)形式同樣落于本申請(qǐng)所附權(quán)利要求書(shū)所限定的范圍。
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210 215 220Pro Lys Asn Leu Gly Ser Pro Leu Asn Ser Ser Glu Ser Glu Ile Leu225 230 235 240Pro Phe Ile His Gln Asp Gly Glu Gln Leu Tyr Phe Ser Ser Asn Arg245 250 255Glu Asp Glu Arg Lys Lys Phe Lys Ile Phe Arg Ile Phe Leu Lys Tyr260 265 270Lys Ser Ala Leu Asp Asn Met Leu Glu Asp Glu Glu Glu Thr Glu Glu275 280 285Thr Pro Thr Thr Lys Pro Thr Glu Ile Leu Ile Pro Lys Ile Asp Gln290 295 300Ser Ser Leu Leu Leu Leu Pro Lys Pro Phe Asn Thr Asp Lys Trp Glu305 310 315 320Gly Phe Asp Asn Glu Gly Ile Ser Phe Asp Lys Asp Gly Ile Trp Ala325 330 335Tyr Ile Ser Ser Asn Arg Ser Gly Gly Glu Gly Gln Phe Asp Ile Phe340 345 350Arg Phe Gln Val Pro Glu Ser Ile Arg Asn Ser Tyr Thr Leu Asn Phe355 360 365Lys Gly Leu Val Leu Asp Gly Ser Glu Lys Thr Met Ile Gly Leu Asp370 375 380Ser Thr Leu Lys Ile Tyr Asp Gly Thr Lys Pro Ala Asn Val Ile Thr385 390 395 400Ser Lys Arg Ile Gly Gly Asp Leu Thr Lys Gly Lys Pro Ser Asn Phe405 410 415Ala Thr Thr Leu Gln Thr Gly Lys Val Tyr Lys Ile Glu Ile Ser Ser420 425 430Pro Gly Phe His Pro Gln Glu Asp Ile Leu Asp Leu Arg Gly Asn Ile435 440 445Gly Lys Asn Arg Lys Val Tyr Arg Thr Tyr Val Leu Leu Pro Ile Gln450 455 460Val Glu Glu Gly Lys Thr Glu Glu Thr Lys Ile Glu Gln Pro Ile Glu465 470 475 480Asn Gln Lys Pro Asn Ser Ala Ala Leu Lys Val Ile Val Ala Asp Ala485 490 495Ser Thr Lys Gln Ile Ile Pro Asp Ala Lys Val Thr Leu Phe Thr Pro500 505 510Met Asn Arg Lys Gly Glu Ser Leu Val Gln Asp Ala Asp Lys Lys Ser515 520 525Phe Leu Ile Lys Lys Leu Pro Asp Asn Asp Phe Glu Leu Phe Ala Thr530 535 540Ala Ser Lys Tyr Ile Ser Glu Ser Ile Asn Ile Ile Gln Lys Asn Ile545 550 555 560Ser Lys Asn Gly Thr Val Thr Ile Tyr Leu Lys Ala Glu Ser Asp Val565 570 575Asp Pro Val Tyr Asn Leu Arg Val Tyr Phe Glu Phe Asn Lys Thr Lys580 585 590Ile Thr Glu Glu Asn Lys Lys Leu Leu Asp Pro Leu Val Gly Tyr Leu595 600 605Leu Lys Asn Ala Ser Asp Lys Ile Glu Ile Gly Gly His Thr Asp Asn610 615 620Val Ala Ser Lys Glu Tyr Asn Thr Arg Leu Ser Ala Lys Arg Ala Arg625 630 635 640Asn Val Tyr Glu Tyr Leu Leu Ser Lys Gly Ile Pro Glu Lys Arg Met645 650 655Arg Ile Arg Ala Tyr Trp Tyr Ser Gln Pro Asp Ala Asp Asn Glu Thr660 665 670Glu Thr Gly Arg Ala Lys Asn Arg Arg Val Gly Phe Arg Lys Leu675 680 685<210>3<211>1428<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>3ttggaaagtt ttttacactt tccttatgtt ttaaaggaat cttacgaaac taaaactatg60gggaaatttt ccaatcaacc ttcaaaatcc aaaatcataa acagacgttt acgaagaatt 120gtatcatacg tttttattat ttctacattg atcggtttta cttctacttt tccagaagaa 180tcggacaaag ttctctttcg ttggaaacta aaaccaggag agaatgtaga attaaacgaa 240tatcatcgag tacaattagt aagtatggga aagaaaatta gaagagaaga taaaaatcga 300atcctattac aaaccctttc ctgcgaaaat aaagaatgta cgttagaagg attttttgat 360acttacactc gttttcccga agttgatcct gcgtttcgta aagataaaac gtttaagagc 420agatttcaaa taacagattt aggtcaatat aaagtgccac aggaatacag tatgcctaat 480ctccgctctc ttcctagttt ttccgaaaaa ccaatttcca ttggagaaga atggacacaa 540cccgctacgg aaagttttca atttccagga ggaagggtta tgatcacggt ttttgcgaaa 600tataagtatc acggtgtaga cgaatgggaa tatcaaaagt tgtccggtaa aggagatcgt 660atcgaataca attataattt atattatgat tctcaaatga acagaaccgg ggttccattt 720aagatctacg gatttgcgcg aggaatggta ttttttgatc gcgaacttgg aattccacaa 780tataaaaggg ttcagttgtc gtacactttt gtctatgaaa atggaatggc gcaagagatg 840tcgttcgaca ttcacggagt atataataaa aacgtaaaac ttacggatca agacaaggac 900aagtttgcgg aagaaatccg taaaatttta ggaggagaac ttccaactgg aattgaacca 960gattccgatt caaaaaagaa tttaaaaaaa ccgaaaaagg aaaccatttc atggccggaa 1020gaagaggata atcaagccgc accagaaagt tcgccggtag aaattagaag aacagaagaa 1080ggaatcgcta tttctttaaa ttcagtgttg tttgatcaca acagctcaga acttaaacaa 1140gaagcaaaat tagaattaaa aagaatcgca tccgtattga aaaaatatgc agatagagaa 1200atcagaatta gcgggcatac tgacaattca ggcggggaag aatataatcg aaaactttcc 1260agagaaagag cactttccgt tttgaaggaa ctcagagatg aacaaggttt ggaagaaaaa 1320agaatgtctt acgaaggata tgggaaatca aaaccgattg cagataactc tacgattcaa 1380ggtaggcaaa aaaatcgtag ggtagatatt actattgtaa tggaataa 1428<210>4<211>475<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>4Met Glu Ser Phe Leu His Phe Pro Tyr Val Leu Lys Glu Ser Tyr Glu1 5 10 15Thr Lys Thr Met Gly Lys Phe Ser Asn Gln Pro Ser Lys Ser Lys Ile20 25 30Ile Asn Arg Arg Leu Arg Arg Ile Val Ser Tyr Val Phe Ile Ile Ser35 40 45Thr Leu Ile Gly Phe Thr Ser Thr Phe Pro Glu Glu Ser Asp Lys Val50 55 60Leu Phe Arg Trp Lys Leu Lys Pro Gly Glu Asn Val Glu Leu Asn Glu65 70 75 80Tyr His Arg Val Gln Leu Val Ser Met Gly Lys Lys Ile Arg Arg Glu85 90 95Asp Lys Asn Arg Ile Leu Leu Gln Thr Leu Ser Cys Glu Asn Lys Glu100 105 110Cys Thr Leu Glu Gly Phe Phe Asp Thr Tyr Thr Arg Phe Pro Glu Val115 120 125Asp Pro Ala Phe Arg Lys Asp Lys Thr Phe Lys Ser Arg Phe Gln Ile130 135 140Thr Asp Leu Gly Gln Tyr Lys Val Pro Gln Glu Tyr Ser Met Pro Asn145 150 155 160Leu Arg Ser Leu Pro Ser Phe Ser Glu Lys Pro Ile Ser Ile Gly Glu165 170 175Glu Trp Thr Gln Pro Ala Thr Glu Ser Phe Gln Phe Pro Gly Gly Arg180 185 190Val Met Ile Thr Val Phe Ala Lys Tyr Lys Tyr His Gly Val Asp Glu195 200 205Trp Glu Tyr Gln Lys Leu Ser Gly Lys Gly Asp Arg Ile Glu Tyr Asn210 215 220Tyr Asn Leu Tyr Tyr Asp Ser Gln Met Asn Arg Thr Gly Val Pro Phe225 230 235 240Lys Ile Tyr Gly Phe Ala Arg Gly Met Val Phe Phe Asp Arg Glu Leu245 250 255Gly Ile Pro Gln Tyr Lys Arg Val Gln Leu Ser Tyr Thr Phe Val Tyr260 265 270Glu Asn Gly Met Ala Gln Glu Met Ser Phe Asp Ile His Gly Val Tyr275 280 285Asn Lys Asn Val Lys Leu Thr Asp Gln Asp Lys Asp Lys Phe Ala Glu290 295 300Glu Ile Arg Lys Ile Leu Gly Gly Glu Leu Pro Thr Gly Ile Glu Pro305 310 315 320Asp Ser Asp Ser Lys Lys Asn Leu Lys Lys Pro Lys Lys Glu Thr Ile325 330 335Ser Trp Pro Glu Glu Glu Asp Asn Gln Ala Ala Pro Glu Ser Ser Pro340 345 350Val Glu Ile Arg Arg Thr Glu Glu Gly Ile Ala Ile Ser Leu Asn Ser355 360 365Val Leu Phe Asp His Asn Ser Ser Glu Leu Lys Gln Glu Ala Lys Leu370 375 380Glu Leu Lys Arg Ile Ala Ser Val Leu Lys Lys Tyr Ala Asp Arg Glu385 390 395 400Ile Arg Ile Ser Gly His Thr Asp Asn Ser Gly Gly Glu Glu Tyr Asn405 410 415Arg Lys Leu Ser Arg Glu Arg Ala Leu Ser Val Leu Lys Glu Leu Arg420 425 430Asp Glu Gln Gly Leu Glu Glu Lys Arg Met Ser Tyr Glu Gly Tyr Gly435 440 445Lys Ser Lys Pro Ile Ala Asp Asn Ser Thr Ile Gln Gly Arg Gln Lys450 455 460Asn Arg Arg Val Asp Ile Thr Ile Val Met Glu465 470 475<210>5<211>2928<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>5ttgattcatt gcaaatatcc gggtggtttt agttttacgt tgaagcgaat attctctcca 60attttaaaag gaattctact tgcgatcctt gtaggacttc tattctattc aggcgaattg 120actcaaatcc tttctaaacg aagtgacttc ttaggaaaaa caatcaaaga agttaaattc 180aaaggaaata aaaatactcc tgacgcagat ctcgaatcta tgattgagat caaagtaggt 240aaaatactta caaagagaat tttagatcga gatttaaaaa atctttttaa ttccggtttt 300ttttattttg tagatattca agcggaagat tttcaggacg gtgttcggat catatttgat 360cttaaagaaa gacctagagt tagagaaatt gaatttgtag gagcagacga agtttttcct 420gcggatttaa gagataaact tcctctcaaa gacaatgaag tgatcacacc tcaaaagatc 480acaaaatcta gagatttaat tcttcaaaaa tacagagacg aaggattttt cctcgcttat 540gtaaaagtgg agctgggtaa accagaccct aaaacaaatc tggttcgagt tcgttttatt 600atagatgaag gagaagagat tccagtttct aagattaatg tttacggaaa cgaatcgatt 660gaaacgtctg aaattctttc tattatggaa atgaaagaag aaggagtttt tgaaggtgga 720aattttaaag aatcttcctt tgaaaaggac aaggatacga ttgttgctta tctcaaaagt 780aaaggttatc tagatgctga attgatccga gagggaacga attgggagat tcactgggaa 840aatccagaaa aaaaagatag aagggtcatt atcgtaaaca ttaaaatttc cgaaggccag 900gtttattttt tcaacggtta tactttaaat catgatatgt ctttagatgg agagggaaga 960cctctctttt taaacaaaga aaaaaaccct cccgaaacta caaaagagga attaaaacct1020ctatttacct ctaaagaaat cgaaaaaagt ttagattaca gcgacgcaga cgtaggtgtg1080atttttgacg aaactcgttt tatgagagat cgtggagcga tgaacgaact ttacagttcg1140agaggttatc tttttgccca ggtaattcct cgtagaaaaa tagtatcttt agatcgtgaa1200aatttagaat attatgaaaa ttgttatagt cgtaaatccg aagaagaaag aagaatctgt1260gaaaacgaat attctcaact tcatgtcaaa cgtttaagac aactttacaa caccaaacct1320gagttacacg gtaaaaaatt cgttcacgtg gactttaata tacgagaaaa caatcttgct1380tatgtggaaa acgtaatcat caaagggaat aaaaaaactc aagatcgtgt aatccgaaga1440gaacttctat ttaaacaagg agatttgttt aattctattt tagtcaatcg ttcccgtgaa1500aggattttta acctaggtta ttttaaagaa gtaaacttta acatgagacc aggttccgat1560cagactaaaa tgaatttgat tatagaagtt gtggaacaac caaccggaac cgtttctatg1620ggaggcggtt atggcacgat cacaggattt tccatcttta cggaagtagg tgaaaataat1680ttaaacggaa ctggtcagaa aatttcaggt cgtttagaat tcggtccttt tagaagactt1740tttcagatta cttggacgga accttggctt tacaataaac cttggtctct ttcactttct1800cttttttatt cttctagaat ttataatgta ggagccgttt cgattaccga aaataataac1860caacaatcca tcaaagaaca agcaatctat tccagagatg gtgtaggttt tacagtagga1920atcggtcata ggatttttat caactggact cattttcata gatattctcc cagtatttac1980gcttctacaa atccttcttc tcttgtatcg gatcaggttc ttgcggaggt tcgtagagga2040tggcaattcc gttctcaaat ttcaaatgga attgcgtatg atattagaga taacgtattt2100aatcctacac aaggttatga cttacttttt cagatagaca atgtagggca ggcgctcggg2160ggacaatctc actttgacca gtatcgagtt cttgcggaat actaccatac ttggttcgat2220tatagctttt ttggactttt tagaaacaat gctctcagac gttggagagt cgttcaagag2280tttagaagtt cttctctttt tacttatcaa agggttccgt attatgggaa acaagatcca2340attcaaaaac cttatattca gttacaagac ttacagttct taggtggata tgaatctcta2400cgcggatggt tttataacga tgcaaagtat cctgccgaat ggagagacgg tgccgctacc2460agaatgatgt tcggttcgga gcttcgtttt ccaattgagc ccaccttact ttggttagtc2520gcttttttcg atgcgggcgc tttatacgaa gaggtaaacc gtgctactgg agtaagaaaa2580gatctttttg aaacttacga ccaaagagtc agagaagctc aaatgaaaga tcccgtgggc2640tattaccttg ctaataatta taatttaacc gctcttagaa aagcggatta tacttttgaa2700gaacttaaca acccggctaa cctggtactt tctgggaata atgttgcttt ggataaattt2760cgattttctt ggggggtagg acttagaatt caaattccgg ttcttccact taggatttat2820tttgcacaaa aattaaagta tactggagtt gcggatcacc ctttcactaa atttgaatct2880gataatgcat ttcagtttgt gtttggaatt ggggactata ggttttag 2928<210>6<211>975<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>6Met Ile His Cys Lys Tyr Pro Gly Gly Phe Ser Phe Thr Leu Lys Arg1 5 10 15Ile Phe Ser Pro Ile Leu Lys Gly Ile Leu Leu Ala Ile Leu Val Gly20 25 30Leu Leu Phe Tyr Ser Gly Glu Leu Thr Gln Ile Leu Ser Lys Arg Ser35 40 45Asp Phe Leu Gly Lys Thr Ile Lys Glu Val Lys Phe Lys Gly Asn Lys50 55 60Asn Thr Pro Asp Ala Asp Leu Glu Ser Met Ile Glu Ile Lys Val Gly65 70 75 80Lys Ile Leu Thr Lys Arg Ile Leu Asp Arg Asp Leu Lys Asn Leu Phe85 90 95Asn Ser Gly Phe Phe Tyr Phe Val Asp Ile Gln Ala Glu Asp Phe Gln100 105 110Asp Gly Val Arg Ile Ile Phe Asp Leu Lys Glu Arg Pro Arg Val Arg115 120 125Glu Ile Glu Phe Val Gly Ala Asp Glu Val Phe Pro Ala Asp Leu Arg130 135 140Asp Lys Leu Pro Leu Lys Asp Asn Glu Val Ile Thr Pro Gln Lys Ile145 150 155 160Thr Lys Ser Arg Asp Leu Ile Leu Gln Lys Tyr Arg Asp Glu Gly Phe165 170 175Phe Leu Ala Tyr Val Lys Val Glu Leu Gly Lys Pro Asp Pro Lys Thr180 185 190Asn Leu Val Arg Val Arg Phe Ile Ile Asp Glu Gly Glu Glu Ile Pro195 200 205Val Ser Lys Ile Asn Val Tyr Gly Asn Glu Ser Ile Glu Thr Ser Glu210 215 220Ile Leu Ser Ile Met Glu Met Lys Glu Glu Gly Val Phe Glu Gly Gly225 230 235 240Asn Phe Lys Glu Ser Ser Phe Glu Lys Asp Lys Asp Thr Ile Val Ala245 250 255Tyr Leu Lys Ser Lys Gly Tyr Leu Asp Ala Glu Leu Ile Arg Glu Gly260 265 270Thr Asn Trp Glu Ile His Trp Glu Asn Pro Glu Lys Lys Asp Arg Arg275 280 285Val Ile Ile Val Asn Ile Lys Ile Ser Glu Gly Gln Val Tyr Phe Phe290 295 300Asn Gly Tyr Thr Leu Asn His Asp Met Ser Leu Asp Gly Glu Gly Arg305 310 315 320Pro Leu Phe Leu Asn Lys Glu Lys Asn Pro Pro Glu Thr Thr Lys Glu325 330 335Glu Leu Lys Pro Leu Phe Thr Ser Lys Glu Ile Glu Lys Ser Leu Asp340 345 350Tyr Ser Asp Ala Asp Val Gly Val Ile Phe Asp Glu Thr Arg Phe Met355 360 365Arg Asp Arg Gly Ala Met Asn Glu Leu Tyr Ser Ser Arg Gly Tyr Leu370 375 380Phe Ala Gln Val Ile Pro Arg Arg Lys Ile Val Ser Leu Asp Arg Glu385 390 395 400Asn Leu Glu Tyr Tyr Glu Asn Cys Tyr Ser Arg Lys Ser Glu Glu Glu405 410 415Arg Arg Ile Cys Glu Asn Glu Tyr Ser Gln Leu His Val Lys Arg Leu420 425 430Arg Gln Leu Tyr Asn Thr Lys Pro Glu Leu His Gly Lys Lys Phe Val435 440 445His Val Asp Phe Asn Ile Arg Glu Asn Asn Leu Ala Tyr Val Glu Asn450 455 460Val Ile Ile Lys Gly Asn Lys Lys Thr Gln Asp Arg Val Ile Arg Arg465 470 475 480Glu Leu Leu Phe Lys Gln Gly Asp Leu Phe Asn Ser Ile Leu Val Asn485 490 495Arg Ser Arg Glu Arg Ile Phe Asn Leu Gly Tyr Phe Lys Glu Val Asn500 505 510Phe Asn Met Arg Pro Gly Ser Asp Gln Thr Lys Met Asn Leu Ile Ile515 520 525Glu Val Val Glu Gln Pro Thr Gly Thr Val Ser Met Gly Gly Gly Tyr530 535 540Gly Thr Ile Thr Gly Phe Ser Ile Phe Thr Glu Val Gly Glu Asn Asn545 550 555 560Leu Asn Gly Thr Gly Gln Lys Ile Ser Gly Arg Leu Glu Phe Gly Pro565 570 575Phe Arg Arg Leu Phe Gln Ile Thr Trp Thr Glu Pro Trp Leu Tyr Asn580 585 590Lys Pro Trp Ser Leu Ser Leu Ser Leu Phe Tyr Ser Ser Arg Ile Tyr595 600 605Asn Val Gly Ala Val Ser Ile Thr Glu Asn Asn Asn Gln Gln Ser Ile610 615 620Lys Glu Gln Ala Ile Tyr Ser Arg Asp Gly Val Gly Phe Thr Val Gly625 630 635 640Ile Gly His Arg Ile Phe Ile Asn Trp Thr His Phe His Arg Tyr Ser645 650 655Pro Ser Ile Tyr Ala Ser Thr Asn Pro Ser Ser Leu Val Ser Asp Gln660 665 670Val Leu Ala Glu Val Arg Arg Gly Trp Gln Phe Arg Ser Gln Ile Ser675 680 685Asn Gly Ile Ala Tyr Asp Ile Arg Asp Asn Val Phe Asn Pro Thr Gln
690 695 700Gly Tyr Asp Leu Leu Phe Gln Ile Asp Asn Val Gly Gln Ala Leu Gly705 710 715 720Gly Gln Ser His Phe Asp Gln Tyr Arg Val Leu Ala Glu Tyr Tyr His725 730 735Thr Trp Phe Asp Tyr Ser Phe Phe Gly Leu Phe Arg Asn Asn Ala Leu740 745 750Arg Arg Trp Arg Val Val Gln Glu Phe Arg Ser Ser Ser Leu Phe Thr755 760 765Tyr Gln Arg Val Pro Tyr Tyr Gly Lys Gln Asp Pro Ile Gln Lys Pro770 775 780Tyr Ile Gln Leu Gln Asp Leu Gln Phe Leu Gly Gly Tyr Glu Ser Leu785 790 795 800Arg Gly Trp Phe Tyr Asn Asp Ala Lys Tyr Pro Ala Glu Trp Arg Asp805 810 815Gly Ala Ala Thr Arg Met Met Phe Gly Ser Glu Leu Arg Phe Pro Ile820 825 830Glu Pro Thr Leu Leu Trp Leu Val Ala Phe Phe Asp Ala Gly Ala Leu835 840 845Tyr Glu Glu Val Asn Arg Ala Thr Gly Val Arg Lys Asp Leu Phe Glu850 855 860Thr Tyr Asp Gln Arg Val Arg Glu Ala Gln Met Lys Asp Pro Val Gly865 870 875 880Tyr Tyr Leu Ala Asn Asn Tyr Asn Leu Thr Ala Leu Arg Lys Ala Asp885 890 895Tyr Thr Phe Glu Glu Leu Asn Asn Pro Ala Asn Leu Val Leu Ser Gly900 905 910Asn Asn Val Ala Leu Asp Lys Phe Arg Phe Ser Trp Gly Val Gly Leu915 920 925Arg Ile Gln Ile Pro Val Leu Pro Leu Arg Ile Tyr Phe Ala Gln Lys930 935 940Leu Lys Tyr Thr Gly Val Ala Asp His Pro Phe Thr Lys Phe Glu Ser945 950 955 960Asp Asn Ala Phe Gln Phe Val Phe Gly Ile Gly Asp Tyr Arg Phe965 970 975<210>7<211>1101<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>7atgagaagaa acataatgaa aattgccgct gtagcagcac ttactgttgc tctaacggca 60tgtaaaggtg acgatgatga tgacgatgtt gttatgttgg cacttttgta tttagcagat120caaacaagcg gaaactgtgt tactcttagc aaggctgacg gtaatggaga tggaatacct 180acttacacag caactggtaa tacgaaacca aaaggagctt gtaacaattt ctataatgta 240actgccattg ttaacaatcc agaagcagtc gtaagttcag ttaaagcatc ctatcaggct 300gctaaggata aaactacagc agctggttca aattgcgctg tagtaagcac ttatcttcaa 360agtgcaattg atgctgtaac aacacttaaa gtacaacaaa ctttagcact tactgcttgt 420tcacgaatcg ggacaggttg gaacctcaat cctttaactt tcggtggtag ttttagtgta 480gatcctgctt atctcggaaa aaccgtttat gcttgttctt ctgaaaaagc aaaagaggaa 540ttatttgact cgtttgctat cgatttctca actgttgctg gatcggttgc aacggatatg 600gcaacgaact tggcatatag acaaaaattg actgcggttt ctgcgtctaa ctttaaatgg 660actgcagatg cagctgctaa aggtcgtttg atcaatgtta ctgagcttac aactgttggt 720aaatccggag caactgtagt tgcatttgga gctattcaat ctgcagcttt agcagcaatt 780ttagctggta acttagctgg agcaaatgca gcagcttctt gtggaaaaga tctgttagct 840aaagaagctg aagatgttca aaaaattgct tttgcacttt atgatccagc gtctggtttt 900aatggtgcga tgacaggtgg aaccttggat agtacaatta ctactgctca ggctcagtct 960gtaactgagg tccttttttc cagccttact tgtcaatatg gtgactttga cgaagaagct 1020gtaggaaata aacaaacagt tggaactgag gcaaacgtaa aaattagtgg aacttgccca 1080acaacttatc ctaaatacta a 1101<210>8<211>366<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>8Met Arg Arg Asn Ile Met Lys Ile Ala Ala Val Ala Ala Leu Thr Val1 5 10 15Ala Leu Thr Ala Cys Lys Gly Asp Asp Asp Asp Asp Asp Val Val Met20 25 30Leu Ala Leu Leu Tyr Leu Ala Asp Gln Thr Ser Gly Asn Cys Val Thr35 40 45Leu Ser Lys Ala Asp Gly Asn Gly Asp Gly Ile Pro Thr Tyr Thr Ala50 55 60Thr Gly Asn Thr Lys Pro Lys Gly Ala Cys Asn Asn Phe Tyr Asn Val65 70 75 80Thr Ala Ile Val Asn Asn Pro Glu Ala Val Val Ser Ser Val Lys Ala85 90 95Ser Tyr Gln Ala Ala Lys Asp Lys Thr Thr Ala Ala Gly Ser Asn Cys100 105 110Ala Val Val Ser Thr Tyr Leu Gln Ser Ala Ile Asp Ala Val Thr Thr115 120 125Leu Lys Val Gln Gln Thr Leu Ala Leu Thr Ala Cys Ser Arg Ile Gly130 135 140Thr Gly Trp Asn Leu Asn Pro Leu Thr Phe Gly Gly Ser Phe Ser Val145 150 155 160Asp Pro Ala Tyr Leu Gly Lys Thr Val Tyr Ala Cys Ser Ser Glu Lys165 170 175Ala Lys Glu Glu Leu Phe Asp Ser Phe Ala Ile Asp Phe Ser Thr Val180 185 190Ala Gly Ser Val Ala Thr Asp Met Ala Thr Asn Leu Ala Tyr Arg Gln195 200 205Lys Leu Thr Ala Val Ser Ala Ser Asn Phe Lys Trp Thr Ala Asp Ala210 215 220Ala Ala Lys Gly Arg Leu Ile Asn Val Thr Glu Leu Thr Thr Val Gly225 230 235 240Lys Ser Gly Ala Thr Val Val Ala Phe Gly Ala Ile Gln Ser Ala Ala245 250 255Leu Ala Ala Ile Leu Ala Gly Asn Leu Ala Gly Ala Asn Ala Ala Ala260 265 270Ser Cys Gly Lys Asp Leu Leu Ala Lys Glu Ala Glu Asp Val Gln Lys275 280 285Ile Ala Phe Ala Leu Tyr Asp Pro Ala Ser Gly Phe Asn Gly Ala Met290 295 300Thr Gly Gly Thr Leu Asp Ser Thr Ile Thr Thr Ala Gln Ala Gln Ser305 310 315 320Val Thr Glu Val Leu Phe Ser Ser Leu Thr Cys Gln Tyr Gly Asp Phe325 330 335Asp Glu Glu Ala Val Gly Asn Lys Gln Thr Val Gly Thr Glu Ala Asn340 345 350Val Lys Ile Ser Gly Thr Cys Pro Thr Thr Tyr Pro Lys Tyr355 360 365<210>9<211>1173<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>9atgggtatga agaagttctt aatcgttttc tcaagcgtat tgaccactgg tcttctcgtg 60tttaacgctt gtaagaaacc taccgaaagt tccaaggcag ccgctactaa aggaaatagt120ccttccgccg ttgttgtgtt tagcgtagga gaagctaaaa ttcttcacgc agatctaacc180gaagaaaaag ctgctttagg tgcaagtttg aaaactggag acaaagtcag taccaaacaa240aagtccaaag ttgatattca atttgcagac ggatctgcaa ttcgaatctc cgaaaattca300gtcatcgatt ttgatgcact ttctatcaat tctcatggaa attccgatac aagacttgca360cttgtttccg gaaaagtttt tgcaaaagtc aacaaggctt cgaaagaaga tcagttttca420gtggtcactc caactgcgat cgctggtgtt agaggaactt cctttatcgt agacagatct480aaatctgaca aagcggtcgt gaaagtttta gacggagcag tcgcagtggc tccacgagtt540gtggttttgg aaggattgag cgatgaagaa attgcaaaga atgaagatct gaaaaaaatt600cagcagaccg ttgcttcttc agagattgtt ttggaaaaaa accaggcttc cgtagtaaaa 660gcggatgaaa aatctttaga tgtaaaagat acttctaaaa tcagtgaaaa gaatattacc 720ggcgttgtga aaaagctgga taattctgga atttctaaga aagaagagga agagatccgt 780acgattgtaa ccgtagataa agacactacg gaaaagatgg ttcggctaaa cgaagaatct 840tcagggaaag ttgacgaaca aaaagcagcc gttttagagg ctgagagaaa aaaattagaa 900agcgaagtag cagctcgtca agaagaagag gctaagaaat tcaaacaagt attgatttct 960gctcctaaag aactgaagtc cagcaaagac atagtaaact attatgaaag aatcgagaag 1020attattatga tggatggatc ttctatgatt ggtgctattg tagatcagca aggatcaaca 1080atgattgttc atacagaaca ggggatcaaa aagatcaatc aagcagatgt tcaagaagta 1140atttatgact tccaaacaaa agctaaattc tga 1173<210>10<211>390<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>10Met Gly Met Lys Lys Phe Leu Ile Val Phe Ser Ser Val Leu Thr Thr1 5 10 15Gly Leu Leu Val Phe Asn Ala Cys Lys Lys Pro Thr Glu Ser Ser Lys20 25 30Ala Ala Ala Thr Lys Gly Asn Ser Pro Ser Ala Val Val Val Phe Ser35 40 45Val Gly Glu Ala Lys Ile Leu His Ala Asp Leu Thr Glu Glu Lys Ala50 55 60Ala Leu Gly Ala Ser Leu Lys Thr Gly Asp Lys Val Ser Thr Lys Gln65 70 75 80Lys Ser Lys Val Asp Ile Gln Phe Ala Asp Gly Ser Ala Ile Arg Ile85 90 95Ser Glu Asn Ser Val Ile Asp Phe Asp Ala Leu Ser Ile Asn Ser His100 105 110Gly Asn Ser Asp Thr Arg Leu Ala Leu Val Ser Gly Lys Val Phe Ala115 120 125Lys Val Asn Lys Ala Ser Lys Glu Asp Gln Phe Ser Val Val Thr Pro130 135 140Thr Ala Ile Ala Gly Val Arg Gly Thr Ser Phe Ile Val Asp Arg Ser145 150 155 160Lys Ser Asp Lys Ala Val Val Lys Val Leu Asp Gly Ala Val Ala Val165 170 175Ala Pro Arg Val Val Val Leu Glu Gly Leu Ser Asp Glu Glu Ile Ala180 185 190Lys Asn Glu Asp Leu Lys Lys Ile Gln Gln Thr Val Ala Ser Ser Glu195 200 205Ile Val Leu Glu Lys Asn Gln Ala Ser Val Val Lys Ala Asp Glu Lys
210 215 220Ser Leu Asp Val Lys Asp Thr Ser Lys Ile Ser Glu Lys Asn Ile Thr225 230 235 240Gly Val Val Lys Lys Leu Asp Asn Ser Gly Ile Ser Lys Lys Glu Glu245 250 255Glu Glu Ile Arg Thr Ile Val Thr Val Asp Lys Asp Thr Thr Glu Lys260 265 270Met Val Arg Leu Asn Glu Glu Ser Ser Gly Lys Val Asp Glu Gln Lys275 280 285Ala Ala Val Leu Glu Ala Glu Arg Lys Lys Leu Glu Ser Glu Val Ala290 295 300Ala Arg Gln Glu Glu Glu Ala Lys Lys Phe Lys Gln Val Leu Ile Ser305 310 315 320Ala Pro Lys Glu Leu Lys Ser Ser Lys Asp Ile Val Asn Tyr Tyr Glu325 330 335Arg Ile Glu Lys Ile Ile Met Met Asp Gly Ser Ser Met Ile Gly Ala340 345 350Ile Val Asp Gln Gln Gly Ser Thr Met Ile Val His Thr Glu Gln Gly355 360 365Ile Lys Lys Ile Asn Gln Ala Asp Val Gln Glu Val Ile Tyr Asp Phe370 375 380Gln Thr Lys Ala Lys Phe385 390<210>11<211>978<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>11ttggtaatct tcggggaaag gaaagtaaac atgagaagat tcatgaactc gttcaaaatc 60actgcggctc taactacgat tctaggagga gtgattctat taggaatcgt gaatggacaa120aacatcatca aaggcaaacg tggaatcgat accgcgacgg gaatcgacgt aagcggtctg180gaactcagat ctatcacggt ggaatcctgg gataatccag cgccttcggc tccttatggt240tgggaagtta ctaccgataa agatacacaa caaccacaag gtggacaaca gcaagcgtat300caagcgactg cccaaaacgc tcaagctcta agagaagtaa aactaatttc tggtaaacct360ggggatataa agaatgtaga cgcagggacc gcaaaagttc tgggggttaa atttcaattc420acttatccag gagagaatgc tgtaacgatt cgtcctcctc gaattcctga atatgaaata480cttcgtacta aagcatactt agatgcaaac aatcagaaaa aagtttctaa gatctatgga540gtggaatttc cgggtgttag taaggcaatg tccgtttggg tttgtggaag aggaaacgag600tataatttag aaggttggat tgaagattgg aaaggagaca cgcacattct tcaatttgga660tctctcgact ttattggatg gagaccattg accgtttaca tccctcaagg agttccacag720gatgtaaatt cttatccgca ggtaaagacg atcgttttca aacagtttaa ggttcgttct780cgtccagata ccagtggaga aactgtatat cttttctttg acgaattgag agtattgtcc840gatgtattcg aagttcactt cgacggagct tctatcgatt tcgatgatga agattgcaga900agcaaacata aattggataa gatgctcaaa accaaagttg aaaaagaatg tggcggcggc960ggtgctgcag gcaaataa 978<210>12<211>325<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>12Met Val Ile Phe Gly Glu Arg Lys Val Asn Met Arg Arg Phe Met Asn1 5 10 15Ser Phe Lys Ile Thr Ala Ala Leu Thr Thr Ile Leu Gly Gly Val Ile20 25 30Leu Leu Gly Ile Val Asn Gly Gln Asn Ile Ile Lys Gly Lys Arg Gly35 40 45Ile Asp Thr Ala Thr Gly Ile Asp Val Ser Gly Leu Glu Leu Arg Ser50 55 60Ile Thr Val Glu Ser Trp Asp Asn Pro Ala Pro Ser Ala Pro Tyr Gly65 70 75 80Trp Glu Val Thr Thr Asp Lys Asp Thr Gln Gln Pro Gln Gly Gly Gln85 90 95Gln Gln Ala Tyr Gln Ala Thr Ala Gln Asn Ala Gln Ala Leu Arg Glu100 105 110Val Lys Leu Ile Ser Gly Lys Pro Gly Asp Ile Lys Asn Val Asp Ala115 120 125Gly Thr Ala Lys Val Leu Gly Val Lys Phe Gln Phe Thr Tyr Pro Gly130 135 140Glu Asn Ala Val Thr Ile Arg Pro Pro Arg Ile Pro Glu Tyr Glu Ile145 150 155 160Leu Arg Thr Lys Ala Tyr Leu Asp Ala Asn Asn Gln Lys Lys Val Ser165 170 175Lys Ile Tyr Gly Val Glu Phe Pro Gly Val Ser Lys Ala Met Ser Val180 185 190Trp Val Cys Gly Arg Gly Asn Glu Tyr Asn Leu Glu Gly Trp Ile Glu195 200 205Asp Trp Lys Gly Asp Thr His Ile Leu Gln Phe Gly Ser Leu Asp Phe210 215 220Ile Gly Trp Arg Pro Leu Thr Val Tyr Ile Pro Gln Gly Val Pro Gln225 230 235 240Asp Val Asn Ser Tyr Pro Gln Val Lys Thr Ile Val Phe Lys Gln Phe245 250 255Lys Val Arg Ser Arg Pro Asp Thr Ser Gly Glu Thr Val Tyr Leu Phe260 265 270Phe Asp Glu Leu Arg Val Leu Ser Asp Val Phe Glu Val His Phe Asp
275 280 285Gly Ala Ser Ile Asp Phe Asp Asp Glu Asp Cys Arg Ser Lys His Lys290 295 300Leu Asp Lys Met Leu Lys Thr Lys Val Glu Lys Glu Cys Gly Gly Gly305 310 315 320Gly Ala Ala Gly Lys325<210>13<211>993<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>13ttgcaatttt ctctcctaca atttgttatt ttaaatgtca tctttttaga gtttgggggt 60tttatgaaac gttatttgag tattgtgatt ctctgtacgt ttgcgatgtt attgctcgtt 120tgtagcacga acaaatcatc cggcagcgat caggtaaaaa ccgagtctaa tgctaccagc 180gcaagaatcg tttggcttct tggagacgta aaaattcttt ccgattctgg agaaaaaaaa 240gcagagttag gtgcttctct ttcttctacg gaccgtgttg taacaggacc gaacggcgga 300gctgagatta tggtcgcaga cagtggaatt attaagatgt ctaaaaattc cgatatagaa 360atttccagcc ttatgaatcc aaacggttcc gatacaaatg ttcaagtgaa ctacggaaag 420atcgtgacga tggtaaaaaa aggtcagaaa actacagagt ttaccgtttc tactccgact 480gcacttgcag gtgttagggg aacttccttc ctaacttctg tagaaagtcc agaaggatct 540aagattaatt gtgccaaagc caattgtacc gttcgttttg cagtgatcga agggaccatt 600gcagtttcta aaaagggaga atcttccgaa gtaattctga gtaaaaaccg cgagctcaga 660attgaaaaga atcaaaaact caccgacaaa ctgattcgtt cacttcaaaa cgattctctt 720tccgaaatga aagaactcat cgttcttcat aaaaacgaaa ctttcgaata cggaaaatta 780gtagaagaac ttaaatcttc cagcgaagaa ctcaaaattt tgagccaatc tggttctgtg 840gaagaagtaa aagctgcgtt tcaaaaaaga gaagcggatc gtaataacgc ggacgagatt 900acaaaaaccg ctaaggcagt aaacgaaacc aaatatgtac aacaagatgt acaaaaggaa 960aaactgaaat tgaatccaaa agaaacattt tag 993<210>14<211>330<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>14Met Gln Phe Ser Leu Leu Gln Phe Val Ile Leu Asn Val Ile Phe Leu1 5 10 15Glu Phe Gly Gly Phe Met Lys Arg Tyr Leu Ser Ile Val Ile Leu Cys20 25 30Thr Phe Ala Met Leu Leu Leu Val Cys Ser Thr Asn Lys Ser Ser Gly35 40 45Ser Asp Gln Val Lys Thr Glu Ser Asn Ala Thr Ser Ala Arg Ile Val50 55 60Trp Leu Leu Gly Asp Val Lys Ile Leu Ser Asp Ser Gly Glu Lys Lys65 70 75 80Ala Glu Leu Gly Ala Ser Leu Ser Ser Thr Asp Arg Val Val Thr Gly85 90 95Pro Asn Gly Gly Ala Glu Ile Met Val Ala Asp Ser Gly Ile Ile Lys100 105 110Met Ser Lys Asn Ser Asp Ile Glu Ile Ser Ser Leu Met Asn Pro Asn115 120 125Gly Ser Asp Thr Asn Val Gln Val Asn Tyr Gly Lys Ile Val Thr Met130 135 140Val Lys Lys Gly Gln Lys Thr Thr Glu Phe Thr Val Ser Thr Pro Thr145 150 155 160Ala Leu Ala Gly Val Arg Gly Thr Ser Phe Leu Thr Ser Val Glu Ser165 170 175Pro Glu Gly Ser Lys Ile Asn Cys Ala Lys Ala Asn Cys Thr Val Arg180 185 190Phe Ala Val Ile Glu Gly Thr Ile Ala Val Ser Lys Lys Gly Glu Ser195 200 205Ser Glu Val Ile Leu Ser Lys Asn Arg Glu Leu Arg Ile Glu Lys Asn210 215 220Gln Lys Leu Thr Asp Lys Leu Ile Arg Ser Leu Gln Asn Asp Ser Leu225 230 235 240Ser Glu Met Lys Glu Leu Ile Val Leu His Lys Asn Glu Thr Phe Glu245 250 255Tyr Gly Lys Leu Val Glu Glu Leu Lys Ser Ser Ser Glu Glu Leu Lys260 265 270Ile Leu Ser Gln Ser Gly Ser Val Glu Glu Val Lys Ala Ala Phe Gln275 280 285Lys Arg Glu Ala Asp Arg Asn Asn Ala Asp Glu Ile Thr Lys Thr Ala290 295 300Lys Ala Val Asn Glu Thr Lys Tyr Val Gln Gln Asp Val Gln Lys Glu305 310 315 320Lys Leu Lys Leu Asn Pro Lys Glu Thr Phe325 330<210>15<211>1770<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>15atgcggaacg aaattcgttt gattgtgacg tctttttttc tttttaattt ctcactttac 60tcaaatactt ttttaaagac gaatacagaa gccttctcac ccaattggga tggaaaaaat120gacgtgctcg agtttagaat ttctaaatcg gcgcttcctc gtctttctga ttgggaactc180gtaatcaaaa attctggaga tgatatagta aaaattttca aagcagatca tagaagaaaa240agaggatttt ctttttttgg actgttacaa gatgatacac aactgtctcc gcttgaaatt300acaattccag aatcgattct atggtttgga gaagactcta aaggattttt actcccggat360ggtgaataca aatatagact tagattggtt actgaaaata atgagaattt attatccgaa420gaaaaaacaa ttcttttaga ttctcattcg cctagttctg aaattggtgt caagacaaga480gttctttttt taaacggaga tcgaaattcc tctcgtatca atatttctca aaaagttttc540ggagaatctt ctgactattt tacgggagaa tttttagatt cggaaggaaa ggtcgtaaag600tcttatacgt ggaagcttaa ggatgtgcct tccgttttga tttgggatgg aacagatttt660tctaataaac agttatctaa tggaatttat acgtatcgat tgatcggaag cgacaaagta720aaaaacgaat ctatttccgt aatttctgat cttacgatcc gtaacgaaat gatcggcgta780gatatgttct ccgattctaa aatttattct tatcagtcgg gaagtttaaa aaattcggca840aggtttacat tttatgtttc ccctaaactt aaatcggatt cttacgaaat tgaagttttt900caaaagaaag ggaacgaaga aaaaaccgtc tatcgtttgc gcgaaaccgg agaacccgga960acggaatgga aatgggattt aaagaatcaa accggtgatt ttgtttcggc tgggacttat 1020ttttgtcgat taacggttca tagtcgatat gaacgttatc aatctatacc ttcttcattt 1080gaaatttcta aagaatcttt caatttagat ttatcccttt cgacaaaaga attttctcca 1140gacaacgatg gaaaaaatga tcttttaaga atatatcttt cacatcaggg aattcctcta 1200caatcttggg aaataacgtt atacgaaata cctccttata cttctctcaa acgtaagatc 1260aaaacttgga atggcgaagg gcaacctggc tccgaaattt tttgggaagg tttggatgaa 1320tcaggagttc gaattggttc cttgagtgaa ttttattttg aatggaaata tacggatgta 1380ttaggaagag aatcgaatgg aaaaggggca gaattcaaaa cgacaattct ggtgttagaa 1440gaagatcatt ccttacgaat ttcgattcct gaatctcaag tagaagcgag atggtggagt 1500ttacctggaa aaatccgttc gttgttaaac gaattcccgg gatataaaat cgaattacaa 1560tctcattctt cacatcaagg agacgaggaa gtcaatcaag taggaaccga agaaagagcc 1620agagaagcac ttgaatattt tttttccaag acggtaccat tcggaagaat tcgttttcgg 1680ggttatggag aaactttacc tttaattcca ggctccggaa aatacgaagc ggataaaaat 1740cagagaatcg atttttatct ttcactttaa1770<210>16<211>589<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>16Met Arg Asn Glu Ile Arg Leu Ile Val Thr Ser Phe Phe Leu Phe Asn1 5 10 15Phe Ser Leu Tyr Ser Asn Thr Phe Leu Lys Thr Asn Thr Glu Ala Phe20 25 30Ser Pro Asn Trp Asp Gly Lys Asn Asp Val Leu Glu Phe Arg Ile Ser35 40 45Lys Ser Ala Leu Pro Arg Leu Ser Asp Trp Glu Leu Val Ile Lys Asn
50 55 60Ser Gly Asp Asp Ile Val Lys Ile Phe Lys Ala Asp His Arg Arg Lys65 70 75 80Arg Gly Phe Ser Phe Phe Gly Leu Leu Gln Asp Asp Thr Gln Leu Ser85 90 95Pro Leu Glu Ile Thr Ile Pro Glu Ser Ile Leu Trp Phe Gly Glu Asp100 105 110Ser Lys Gly Phe Leu Leu Pro Asp Gly Glu Tyr Lys Tyr Arg Leu Arg115 120 125Leu Val Thr Glu Asn Asn Glu Asn Leu Leu Ser Glu Glu Lys Thr Ile130 135 140Leu Leu Asp Ser His Ser Pro Ser Ser Glu Ile Gly Val Lys Thr Arg145 150 155 160Val Leu Phe Leu Asn Gly Asp Arg Asn Ser Ser Arg Ile Asn Ile Ser165 170 175Gln Lys Val Phe Gly Glu Ser Ser Asp Tyr Phe Thr Gly Glu Phe Leu180 185 190Asp Ser Glu Gly Lys Val Val Lys Ser Tyr Thr Trp Lys Leu Lys Asp195 200 205Val Pro Ser Val Leu Ile Trp Asp Gly Thr Asp Phe Ser Asn Lys Gln210 215 220Leu Ser Asn Gly Ile Tyr Thr Tyr Arg Leu Ile Gly Ser Asp Lys Val225 230 235 240Lys Asn Glu Ser Ile Ser Val Ile Ser Asp Leu Thr Ile Arg Asn Glu245 250 255Met Ile Gly Val Asp Met Phe Ser Asp Ser Lys Ile Tyr Ser Tyr Gln260 265 270Ser Gly Ser Leu Lys Asn Ser Ala Arg Phe Thr Phe Tyr Val Ser Pro275 280 285Lys Leu Lys Ser Asp Ser Tyr Glu Ile Glu Val Phe Gln Lys Lys Gly290 295 300Asn Glu Glu Lys Thr Val Tyr Arg Leu Arg Glu Thr Gly Glu Pro Gly305 310 315 320Thr Glu Trp Lys Trp Asp Leu Lys Asn Gln Thr Gly Asp Phe Val Ser325 330 335Ala Gly Thr Tyr Phe Cys Arg Leu Thr Val His Ser Arg Tyr Glu Arg340 345 350Tyr Gln Ser Ile Pro Ser Ser Phe Glu Ile Ser Lys Glu Ser Phe Asn355 360 365Leu Asp Leu Ser Leu Ser Thr Lys Glu Phe Ser Pro Asp Asn Asp Gly370 375 380Lys Asn Asp Leu Leu Arg Ile Tyr Leu Ser His Gln Gly Ile Pro Leu385 390 395 400Gln Ser Trp Glu Ile Thr Leu Tyr Glu Ile Pro Pro Tyr Thr Ser Leu405 410 415Lys Arg Lys Ile Lys Thr Trp Asn Gly Glu Gly Gln Pro Gly Ser Glu420 425 430Ile Phe Trp Glu Gly Leu Asp Glu Ser Gly Val Arg Ile Gly Ser Leu435 440 445Ser Glu Phe Tyr Phe Glu Trp Lys Tyr Thr Asp Val Leu Gly Arg Glu450 455 460Ser Asn Gly Lys Gly Ala Glu Phe Lys Thr Thr Ile Leu Val Leu Glu465 470 475 480Glu Asp His Ser Leu Arg Ile Ser Ile Pro Glu Ser Gln Val Glu Ala485 490 495Arg Trp Trp Ser Leu Pro Gly Lys Ile Arg Ser Leu Leu Asn Glu Phe500 505 510Pro Gly Tyr Lys Ile Glu Leu Gln Ser His Ser Ser His Gln Gly Asp515 520 525Glu Glu Val Asn Gln Val Gly Thr Glu Glu Arg Ala Arg Glu Ala Leu530 535 540Glu Tyr Phe Phe Ser Lys Thr Val Pro Phe Gly Arg Ile Arg Phe Arg545 550 555 560Gly Tyr Gly Glu Thr Leu Pro Leu Ile Pro Gly Ser Gly Lys Tyr Glu565 570 575Ala Asp Lys Asn Gln Arg Ile Asp Phe Tyr Leu Ser Leu580 585<210>17<211>1269<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>17atgaacctaa aaaggatttt tatattcttt cttcttcttt gtcaattaca ctgtgctacg 60tttacgggaa cacttgtcaa taaatgggta aaagattatt acgagagcac taatattacc120ggagttggca ttggtttggc tggagatgga gcgattagtg tttatatggg agttcaattt180ggattagcct tcggacttgc ctatgccgct gctacgatcg tagctcttcc ttttattttt240ttcggacttt tacaaccttt gaaagttcag cctgtacttt attttccaat attttcaaac300gacggtaagg atttgagagg tcttttacag catgttccgc acgtaattcc gttttatatt360cgacccatcg taatcccttc tcatccgaaa gaatcgatcg aaagagaatt ggctaaaagt420tttttttcat ccgtgtattt caattttgac tttctttatt cgtttcaata tctcaattca480atctctactt ctaaaatttt agaacatact tcgattgagg aaattcgaaa tgatatacct540cgatttagaa aattattcaa tgaagacttc gtatttattt ccgaagcgcg tctgaaattc600ggagaatgta aattagaaaa aatttcttat aaagaaatca tgaagttcga ggaacgtctt660ttaagtagac ccgtctatcc tagagagaag gaagatcaag aggagaccgg gctgatggaa720gttcaggtgg aaattaaaac gtatttagtt gattttaaaa accagaagat aagatcgttt780gaaatttctg aaaccgaaag aggatataat gatttcgggc aacgtgcctg tcccgatcca840atccgagcgg gaaaaatggc tttaaagaac gtaggtcttt ctcatacaag aaaaatcttt900ccacaatgga agaagacgaa actgaatttg atccgaaacg attctgatcc gaatgtgcag 960aaattattgg aggaaggctt ccagtatttt ggaagaggag acgatgagaa ccgcgaaatc 1020tttttaataa aaccggaagt gttagaggtt tggaaactcg ccttggccca atccggaaat 1080cgatccgagg gcgcgtttgc aaacatagct tactattatc taaacaacgg ttatttagaa 1140gaggcgatcg attggtttcg gaaggccgcg gttgcaaatc cggatagaga aaaattttgg 1200aatctaagaa tcaaagatat cttacacaag agagaacaag tcgaagtgta taatgatctt 1260aacaattaa 1269<210>18<211>422<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>18Met Asn Leu Lys Arg Ile Phe Ile Phe Phe Leu Leu Leu Cys Gln Leu1 5 10 15His Cys Ala Thr Phe Thr Gly Thr Leu Val Asn Lys Trp Val Lys Asp20 25 30Tyr Tyr Glu Ser Thr Asn Ile Thr Gly Val Gly Ile Gly Leu Ala Gly35 40 45Asp Gly Ala Ile Ser Val Tyr Met Gly Val Gln Phe Gly Leu Ala Phe50 55 60Gly Leu Ala Tyr Ala Ala Ala Thr Ile Val Ala Leu Pro Phe Ile Phe65 70 75 80Phe Gly Leu Leu Gln Pro Leu Lys Val Gln Pro Val Leu Tyr Phe Pro85 90 95Ile Phe Ser Asn Asp Gly Lys Asp Leu Arg Gly Leu Leu Gln His Val100 105 110Pro His Val Ile Pro Phe Tyr Ile Arg Pro Ile Val Ile Pro Ser His115 120 125Pro Lys Glu Ser Ile Glu Arg Glu Leu Ala Lys Ser Phe Phe Ser Ser130 135 140Val Tyr Phe Asn Phe Asp Phe Leu Tyr Ser Phe Gln Tyr Leu Asn Ser145 150 155 160Ile Ser Thr Ser Lys Ile Leu Glu His Thr Ser Ile Glu Glu Ile Arg165 170 175Asn Asp Ile Pro Arg Phe Arg Lys Leu Phe Asn Glu Asp Phe Val Phe180 185 190Ile Ser Glu Ala Arg Leu Lys Phe Gly Glu Cys Lys Leu Glu Lys Ilel95 200 205Ser Tyr Lys Glu Ile Met Lys Phe Glu Glu Arg Leu Leu Ser Arg Pro210 215 220Val Tyr Pro Arg Glu Lys Glu Asp Gln Glu Glu Thr Gly Leu Met Glu225 230 235 240Val Gln Val Glu Ile Lys Thr Tyr Leu Val Asp Phe Lys Asn Gln Lys245 250 255Ile Arg Ser Phe Glu Ile Ser Glu Thr Glu Arg Gly Tyr Asn Asp Phe260 265 270Gly Gln Arg Ala Cys Pro Asp Pro Ile Arg Ala Gly Lys Met Ala Leu275 280 285Lys Asn Val Gly Leu Ser His Thr Arg Lys Ile Phe Pro Gln Trp Lys290 295 300Lys Thr Lys Leu Asn Leu Ile Arg Asn Asp Ser Asp Pro Asn Val Gln305 310 315 320Lys Leu Leu Glu Glu Gly Phe Gln Tyr Phe Gly Arg Gly Asp Asp Glu325 330 335Asn Arg Glu Ile Phe Leu Ile Lys Pro Glu Val Leu Glu Val Trp Lys340 345 350Leu Ala Leu Ala Gln Ser Gly Asn Arg Ser Glu Gly Ala Phe Ala Asn355 360 365Ile Ala Tyr Tyr Tyr Leu Asn Asn Gly Tyr Leu Glu Glu Ala Ile Asp370 375 380Trp Phe Arg Lys Ala Ala Val Ala Asn Pro Asp Arg Glu Lys Phe Trp385 390 395 400Asn Leu Arg Ile Lys Asp Ile Leu His Lys Arg Glu Gln Val Glu Val405 410 415Tyr Asn Asp Leu Asn Asn420<210>19<211>1881<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>19tggattccta aagaaaattt tactcaactc gcatcacaaa gagaaaaatt caaaaacgtt 60tctcataacg aaacattaaa actagaaatt ggctataaat tattaactgg aaaaattctt120cagtttggaa aattacttag taccgaaaac gcatacgtcc ccatcgaatc caacgaaatt180acttccgaac tttctcaact caacgacaag accgtaagaa ttttatgcag tatgaaaggt240tccacctgca atcctatacg ttatgaaatt tatccctttt ggtattcgaa agaaatcaaa300ccttggacga tcaaaaaaat tcctgattac gtaaatcata atatttttgc attcaatcct360acagtttccc cagacggtaa atatcttttt tggaccgctt atgtcaaacg aggtaaatcc420ggaactcaaa agatttggta ttccaaatta gacgaaaaag gtttttggga agatggtaag480gaaatgaacg ctcctttaaa taatgaaatg ccttccgctg ttatctctgc acttcctggt540ggtaacgaac ttttcgtctt tggaacgttt ggcgaaaaag aacttttaga cgaactcagt600aaagattttg aaacgaaggc agacttggcc gctcgttctt ctaaaaattc aaacgtatat660agaaaaaaga tagaagaact tagagcagaa tatgacgaaa aaacgaaaca aatttcaagc720agagttcctc tttataaaag ttttaaggaa aaagattcct ggtctaaacc aagcatatta780aactttccta atttttataa tctttataga aagagaaacg attcaagcca agaaattttc 840ggtggatcta ctctttcttc ttcagggaga attttaatct attcttctca acataaggat 900tccaaaggaa aattggatct ttatgttagc aaaatgttaa acgacggaac ctttccttta 960ggtacaaact taggagatgt gatcaacaca actcatgaag agatggctcc ttttttagca 1020agcgatgata gaacccttta tttttctagc gacggtcata aagggctctc tatttatatg 1080acaaaaagaa tcggagaagg ttgggatcaa tggaccaaac ctgtcgaggt ttccgaaaat 1140ctaaaaggtg taaatttttt ttcaattccc gcgaatagcg actgggccta tataagcaaa 1200gacggccgat tgtttatggc ttatcttcct aaagaaatgc gcccagagaa ggtcgtgatc 1260ataaacggaa aagttttaga tacggatggt aaccctcttt ctgctgacat acattatgaa 1320tctttaaagt ctcacgaaaa gatcggtagc gccaaaagcg atccttctaa tggtaatttt 1380tcgatcattc tccccttcgg cgaaaactac ggtttttacg cccagaaaaa aggttatctt 1440ccagtatcac aaaatttgaa tctaagttct aaaaagaaat tctctgaaaa agtggaagta 1500attttacaac ttcctccgat ccgagaaaga ggttccattc aaatcaataa tttatttttt 1560gagtccaaga gttttcaaat cgctccggaa tccgccccag aactggatcg tctcgcggaa 1620atcgtaaagg aaaatccaga tattgaaatt cagattgaag gtcataccga caacattggc 1680aagaaaaaag acaatctaat tctttccgaa aaaagagcgg cagcggtcgc agaatatctt 1740tttcaaaaac attctatttc taagactaga atccaaacca aaggttttgg ggacagtgtt 1800cctttgagta aaaatgattc cgaagaagca cgcaaaaaaa acagaagggt caattttacg 1860attttgaaaa aaagtaaata a 188l<210>20<211>626<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>20Trp Ile Pro Lys Glu Asn Phe Thr Gln Leu Ala Ser Gln Arg Glu Lys1 5 10 15Phe Lys Asn Val Ser His Asn Glu Thr Leu Lys Leu Glu Ile Gly Tyr20 25 30Lys Leu Leu Thr Gly Lys Ile Leu Gln Phe Gly Lys Leu Leu Ser Thr35 40 45Glu Asn Ala Tyr Val Pro Ile Glu Ser Asn Glu Ile Thr Ser Glu Leu50 55 60Ser Gln Leu Asn Asp Lys Thr Val Arg Ile Leu Cys Ser Met Lys Gly65 70 75 80Ser Thr Cys Asn Pro Ile Arg Tyr Glu Ile Tyr Pro Phe Trp Tyr Ser85 90 95Lys Glu Ile Lys Pro Trp Thr Ile Lys Lys Ile Pro Asp Tyr Val Asn100 105 110His Asn Ile Phe Ala Phe Asn Pro Thr Val Ser Pro Asp Gly Lys Tyr115 120 125Leu Phe Trp Thr Ala Tyr Val Lys Arg Gly Lys Ser Gly Thr Gln Lys
130 135 140Ile Trp Tyr Ser Lys Leu Asp Glu Lys Gly Phe Trp Glu Asp Gly Lys145 150 155 160Glu Met Asn Ala Pro Leu Asn Asn Glu Met Pro Ser Ala Val Ile Ser165 170 175Ala Leu Pro Gly Gly Asn Glu Leu Phe Val Phe Gly Thr Phe Gly Glu180 185 190Lys Glu Leu Leu Asp Glu Leu Ser Lys Asp Phe Glu Thr Lys Ala Asp195 200 205Leu Ala Ala Arg Ser Ser Lys Asn Ser Asn Val Tyr Arg Lys Lys Ile210 215 220Glu Glu Leu Arg Ala Glu Tyr Asp Glu Lys Thr Lys Gln Ile Ser Ser225 230 235 240Arg Val Pro Leu Tyr Lys Ser Phe Lys Glu Lys Asp Ser Trp Ser Lys245 250 255Pro Ser Ile Leu Asn Phe Pro Asn Phe Tyr Asn Leu Tyr Arg Lys Arg260 265 270Asn Asp Ser Ser Gln Glu Ile Phe Gly Gly Ser Thr Leu Ser Ser Ser275 280 285Gly Arg Ile Leu Ile Tyr Ser Ser Gln His Lys Asp Ser Lys Gly Lys290 295 300Leu Asp Leu Tyr Val Ser Lys Met Leu Asn Asp Gly Thr Phe Pro Leu305 310 315 320Gly Thr Asn Leu Gly Asp Val Ile Asn Thr Thr His Glu Glu Met Ala325 330 335Pro Phe Leu Ala Ser Asp Asp Arg Thr Leu Tyr Phe Ser Ser Asp Gly340 345 350His Lys Gly Leu Ser Ile Tyr Met Thr Lys Arg Ile Gly Glu Gly Trp355 360 365Asp Gln Trp Thr Lys Pro Val Glu Val Ser Glu Asn Leu Lys Gly Val370 375 380Asn Phe Phe Ser Ile Pro Ala Asn Ser Asp Trp Ala Tyr Ile Ser Lys385 390 395 400Asp Gly Arg Leu Phe Met Ala Tyr Leu Pro Lys Glu Met Arg Pro Glu405 410 415Lys Val Val Ile Ile Asn Gly Lys Val Leu Asp Thr Asp Gly Asn Pro420 425 430Leu Ser Ala Asp Ile His Tyr Glu Ser Leu Lys Ser His Glu Lys Ile435 440 445Gly Ser Ala Lys Ser Asp Pro Ser Asn Gly Asn Phe Ser Ile Ile Leu450 455 460Pro Phe Gly Glu Asn Tyr Gly Phe Tyr Ala Gln Lys Lys Gly Tyr Leu465 470 475 480Pro Val Ser Gln Asn Leu Asn Leu Ser Ser Lys Lys Lys Phe Ser Glu485 490 495Lys Val Glu Val Ile Leu Gln Leu Pro Pro Ile Arg Glu Arg Gly Ser500 505 510Ile Gln Ile Asn Asn Leu Phe Phe Glu Ser Lys Ser Phe Gln Ile Ala515 520 525Pro Glu Ser Ala Pro Glu Leu Asp Arg Leu Ala Glu Ile Val Lys Glu530 535 540Asn Pro Asp Ile Glu Ile Gln Ile Glu Gly His Thr Asp Asn Ile Gly545 550 555 560Lys Lys Lys Asp Asn Leu Ile Leu Ser Glu Lys Arg Ala Ala Ala Val565 570 575Ala Glu Tyr Leu Phe Gln Lys His Ser Ile Ser Lys Thr Arg Ile Gln580 585 590Thr Lys Gly Phe Gly Asp Ser Val Pro Leu Ser Lys Asn Asp Ser Glu595 600 605Glu Ala Arg Lys Lys Asn Arg Arg Val Asn Phe Thr Ile Leu Lys Lys610 615 620Ser Lys625<210>21<211>1287<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>21atgagcctaa aaaataaaaa ttatgtttta tcaaaaaaaa ccatattgat tctattttta60gtatattttg tttttataat ttcttttttt tctatttact ctcaggattt aagtataaat 120caaaatccta aaccagaaaa gttaaaaggt tctatcaata cgagtctcaa tgagtttgga 180atcagtctta ctgatgacgg aaatatttta tatttctatt ctaaaagaca aaattcaaat 240tatacagaca tttataagtc aactcgaacg aaagatgaat ggacacaagg agaggaaatt 300gaagttctaa attcaaactt tgacgaccaa agtcctttca ttttaaaccg agaagaagga 360attctttttt catctaatag agatggtgcg accgaattcc aatttgcaaa tggaaaaatc 420ggagtttcta gagatattta tttttctaaa aaaataaatt cttcttggac agaaccggtt 480cttcttccta gagccgtgaa tacggaagaa atcgaagaaa atccgtttct atttaataat 540agattgtatt ttacccgtta tccttttggg caagtttcag aagcggacat tttcgtttct 600gtttataaaa ataacacttg ggaaaaagca atgagcctcc ctgatccgat taacaccgtt 660tattcggaaa ttgcggctac aattagtaaa gatggaaaga cgatttattt ttcttctaac 720cgtccggggg gttttggcgg ttatgatttg tataagtcta ctttacttga aaacggaaat 780tattccgaac cgattaatct tggacccgaa attaacacta ccggagatga ggcttttttt 840ctggaaacaa acgatagaaa gacattctat ttttgtagaa ggaaagaacg cgattatgat 900atttattcta tcgtttctaa cccgtttcaa gaactagaaa aaggaaaatc tatttctttg 960gatagtatcc atttttcttt gggctcttat gaaattctcg aaaattcttt ttcaatttta 1020gataatttga attcttatct taaggaaaat ttaaatataa aaatcaaaat caccggccat 1080accgatctta atggagattc ccaggacaac cttattctca gccgtaatcg tgcaaatgca 1140gtaaaggatt atttagttaa aaggggaatc gattctcaaa gaattatcac ggatgggaaa 1200ggtagttcgg agccaattgt tcctatgaaa aatccagaga cggattataa aaatagaaga 1260accgaatttc agattatcag tcgttag 1287<210>22<211>428<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>22Met Ser Leu Lys Asn Lys Asn Tyr Val Leu Ser Lys Lys Thr Ile Leu1 5 10 15Ile Leu Phe Leu Val Tyr Phe Val Phe Ile Ile Ser Phe Phe Ser Ile20 25 30Tyr Ser Gln Asp Leu Ser Ile Asn Gln Asn Pro Lys Pro Glu Lys Leu35 40 45Lys Gly Ser Ile Asn Thr Ser Leu Asn Glu Phe Gly Ile Ser Leu Thr50 55 60Asp Asp Gly Asn Ile Leu Tyr Phe Tyr Ser Lys Arg Gln Asn Ser Asn65 70 75 80Tyr Thr Asp Ile Tyr Lys Ser Thr Arg Thr Lys Asp Glu Trp Thr Gln85 90 95Gly Glu Glu Ile Glu Val Leu Asn Ser Asn Phe Asp Asp Gln Ser Pro100 105 110Phe Ile Leu Asn Arg Glu Glu Gly Ile Leu Phe Ser Ser Asn Arg Asp115 120 125Gly Ala Thr Glu Phe Gln Phe Ala Asn Gly Lys Ile Gly Val Ser Arg130 135 140Asp Ile Tyr Phe Ser Lys Lys Ile Asn Ser Ser Trp Thr Glu Pro Val145 150 155 160Leu Leu Pro Arg Ala Val Asn Thr Glu Glu Ile Glu Glu Asn Pro Phe165 170 175Leu Phe Asn Asn Arg Leu Tyr Phe Thr Arg Tyr Pro Phe Gly Gln Val180 185 190Ser Glu Ala Asp Ile Phe Val Ser Val Tyr Lys Asn Asn Thr Trp Glu195 200 205Lys Ala Met Ser Leu Pro Asp Pro Ile Asn Thr Val Tyr Ser Glu Ile210 215 220Ala Ala Thr Ile Ser Lys Asp Gly Lys Thr Ile Tyr Phe Ser Ser Asn225 230 235 240Arg Pro Gly Gly Phe Gly Gly Tyr Asp Leu Tyr Lys Ser Thr Leu Leu245 250 255Glu Asn Gly Asn Tyr Ser Glu Pro Ile Asn Leu Gly Pro Glu Ile Asn260 265 270Thr Thr Gly Asp Glu Ala Phe Phe Leu Glu Thr Asn Asp Arg Lys Thr275 280 285Phe Tyr Phe Cys Arg Arg Lys Glu Arg Asp Tyr Asp Ile Tyr Ser Ile290 295 300Val Ser Asn Pro Phe Gln Glu Leu Glu Lys Gly Lys Ser Ile Ser Leu305 310 315 320Asp Ser Ile His Phe Ser Leu Gly Ser Tyr Glu Ile Leu Glu Asn Ser325 330 335Phe Ser Ile Leu Asp Asn Leu Asn Ser Tyr Leu Lys Glu Asn Leu Asn340 345 350Ile Lys Ile Lys Ile Thr Gly His Thr Asp Leu Asn Gly Asp Ser Gln355 360 365Asp Asn Leu Ile Leu Ser Arg Asn Arg Ala Asn Ala Val Lys Asp Tyr370 375 380Leu Val Lys Arg Gly Ile Asp Ser Gln Arg Ile Ile Thr Asp Gly Lys385 390 395 400Gly Ser Ser Glu Pro Ile Val Pro Met Lys Asn Pro Glu Thr Asp Tyr405 410 415Lys Asn Arg Arg Thr Glu Phe Gln Ile Ile Ser Arg420 425<210>23<211>1092<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>23atggaagaac cgggaatgaa aagggaacaa aagattcaag aaattctttt cgaaggaaaa 60caaaacgatc ttagtccttc aatagaatgg ctggctaaag gtttgagcaa ttcttgggta 120gaatattcac cccaaaaatc agacttcgaa tctctttatg cccaggccag taaaaattct 180tctaaaatac tttccttttc cgattttaca aaaaataaac tcatttgggg aatttcagcg 240gcagcgatat ttctttttgc agttacatta ggatattatt ctattcttaa ggataagccc 300tctctcggag ttgaaaaagg gggggtagaa atttcacaag tagaaggaga agcctacctc 360acctcctccg atccaaaaga caaaattctt ttaaaaccag gggtacgaat ccaagaaggc 420cagagagtag tcactggttc taaagccatt ttgaacttaa aagtgtccga tggaattgta 480gtaagagtac tttctgattc agaagtctcc tttcgattaa ttgatctttc cacccattac 540aaaattggaa tcgatttaga aaagggagaa ttattagctc atattcataa aaatcttaaa 600aaagaagaat ttatagttcg ttccgaaaat ttaagcgcgg aagtaagagg tacaagtttt 660agttttcaga atattcctgg acaaggaacc aaagtagaag ttttagaagg tcgagtagca 720gttagcactc acgtagaatc tcaaaaatca gctcccgaag gtgaacaagt tatagagcca 780aatcagggaa tttttgtaaa tcaaaagggt tttgttcgca gtcatttaaa cgatttcgaa 840aaaaatcgtt tagaaaccga attcgaaaaa ctgccaattg aaaacattcc tcgagataaa 900aaccgaacct attcaaacag acaagagtta ctaacggact tccaaagatt agaaaaaatt 960attttgatca atggaaaaga gatcgaaggt gtaatcgtag acatggatga aaaagcgatg 1020tacattcaaa ctcttgaaaa agaaattgtt attcagagag aatccgtctc tgaagtaatt 1080caactctact ga 1092<210>24<211>363<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>24Met Glu Glu Pro Gly Met Lys Arg Glu Gln Lys Ile Gln Glu Ile Leu1 5 10 15Phe Glu Gly Lys Gln Asn Asp Leu Ser Pro Ser Ile Glu Trp Leu Ala20 25 30Lys Gly Leu Ser Asn Ser Trp Val Glu Tyr Ser Pro Gln Lys Ser Asp35 40 45Phe Glu Ser Leu Tyr Ala Gln Ala Ser Lys Asn Ser Ser Lys Ile Leu50 55 60Ser Phe Ser Asp Phe Thr Lys Asn Lys Leu Ile Trp Gly Ile Ser Ala65 70 75 80Ala Ala Ile Phe Leu Phe Ala Val Thr Leu Gly Tyr Tyr Ser Ile Leu85 90 95Lys Asp Lys Pro Ser Leu Gly Val Glu Lys Gly Gly Val Glu Ile Ser100 105 110Gln Val Glu Gly Glu Ala Tyr Leu Thr Ser Ser Asp Pro Lys Asp Lys115 120 125Ile Leu Leu Lys Pro Gly Val Arg Ile Gln Glu Gly Gln Arg Val Val130 135 140Thr Gly Ser Lys Ala Ile Leu Asn Leu Lys Val Ser Asp Gly Ile Val145 150 155 160Val Arg Val Leu Ser Asp Ser Glu Val Ser Phe Arg Leu Ile Asp Leu165 170 175Ser Thr His Tyr Lys Ile Gly Ile Asp Leu Glu Lys Gly Glu Leu Leu180 185 190Ala His Ile His Lys Asn Leu Lys Lys Glu Glu Phe Ile Val Arg Ser195 200 205Glu Asn Leu Ser Ala Glu Val Arg Gly Thr Ser Phe Ser Phe Gln Asn210 215 220Ile Pro Gly Gln Gly Thr Lys Val Glu Val Leu Glu Gly Arg Val Ala225 230 235 240Val Ser Thr His Val Glu Ser Gln Lys Ser Ala Pro Glu Gly Glu Gln245 250 255Val Ile Glu Pro Asn Gln Gly Ile Phe Val Asn Gln Lys Gly Phe Val260 265 270Arg Ser His Leu Asn Asp Phe Glu Lys Asn Arg Leu Glu Thr Glu Phe275 280 285Glu Lys Leu Pro Ile Glu Asn Ile Pro Arg Asp Lys Asn Arg Thr Tyr290 295 300Ser Asn Arg Gln Glu Leu Leu Thr Asp Phe Gln Arg Leu Glu Lys Ile305 310 315 320Ile Leu Ile Asn Gly Lys Glu Ile Glu Gly Val Ile Val Asp Met Asp325 330 335Glu Lys Ala Met Tyr Ile Gln Thr Leu Glu Lys Glu Ile Val Ile Gln340 345 350Arg Glu Ser Val Ser Glu Val Ile Gln Leu Tyr355 360<210>25<211>1152<212>DNA<213>鉤端螺旋體(Leptospira interrogans)<400>25atggcgaaga aagaaaacta ctatattact atcaaaggta gaaaatatga tcgtaagttg 60atccagctcg cggaagagtt cacttccggt aaacgggacg gtaagatttc gatcaacgac120gcaaaacgtc ttttaaaaat tgtcaaggat aacaacgctt atacggatat agaaaaacat180acgatcgaat acattcgtga aaactataag tttaccgaaa aatcggacga atggttccgt240tcagaaatcc gtaaatgggc cgctaaaaaa gtgcaagaag caaaaaagaa aagtgatgtg300gaatccatcc tagtcgatga ttctgaagcg ccggaaataa attttccttc cagttgggga360gaagacaaaa ccgaagttgt tgaaattaca caaacctcta aaatagattg gagagaaaat420tcaaattttt catccgcaac gtctcactct aaaaaaaata aaaagatcat tccaacttta480atttttcttt ctggtttttt gattcttgta ggtttagttt atttttttcg aaccttattt540tataaggaag acttggaaca agtagtaaaa acgaattctg agattgtctc taattcaaaa600gaaaaacaat ccgacgtttc gatagaaaaa gcagaatcta caaaggaagt tcgaaagaaa660aacgtaagat ctaaaaaaga agaatcggaa attccaaaaa atgctcttac aattctaaaa720cctcaaaccg gaaagaagtt agaatctaaa tccttattct cttcgttgac aaatcaaaat780tccacagaag aattttcttc caatcctcaa tttagagaaa ttgaatccaa tgtaattcgt840tttgaaaaaa acagcattca aattcataaa gaatcgagac caagtctcaa ccgtttggct900cgctggatga aacaggattc ttcgatccga gtcaaagtta tcggtcatac ttctttagag960ggtagcgaag acgccaatca aaaagtttct cttcttcgtg cacaaacggt tcgaaattat 1020atcgccggga atggcatttc caaagatcgt tttgagatta ttcccaaagg cgcaagcgtt 1080cctattggcg ataattctaa agaagagggg aaggaaatga atcgtagagt ggaacttaga 1140atctataatt ga 1152<210>26<211>383<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>26Met Ala Lys Lys Glu Asn Tyr Tyr Ile Thr Ile Lys Gly Arg Lys Tyr1 5 10 15Asp Arg Lys Leu Ile Gln Leu Ala Glu Glu Phe Thr Ser Gly Lys Arg20 25 30Asp Gly Lys Ile Ser Ile Asn Asp Ala Lys Arg Leu Leu Lys Ile Val35 40 45Lys Asp Asn Asn Ala Tyr Thr Asp Ile Glu Lys His Thr Ile Glu Tyr50 55 60Ile Arg Glu Asn Tyr Lys Phe Thr Glu Lys Ser Asp Glu Trp Phe Arg65 70 75 80Ser Glu Ile Arg Lys Trp Ala Ala Lys Lys Val Gln Glu Ala Lys Lys85 90 95Lys Ser Asp Val Glu Ser Ile Leu Val Asp Asp Ser Glu Ala Pro Glu100 105 110Ile Asn Phe Pro Ser Ser Trp Gly Glu Asp Lys Thr Glu Val Val Glu115 120 125Ile Thr Gln Thr Ser Lys Ile Asp Trp Arg Glu Asn Ser Asn Phe Ser130 135 140Ser Ala Thr Ser His Ser Lys Lys Asn Lys Lys Ile Ile Pro Thr Leu145 150 155 160Ile Phe Leu Ser Gly Phe Leu Ile Leu Val Gly Leu Val Tyr Phe Phe165 170 175Arg Thr Leu Phe Tyr Lys Glu Asp Leu Glu Gln Val Val Lys Thr Asn180 185 190Ser Glu Ile Val Ser Asn Ser Lys Glu Lys Gln Ser Asp Val Ser Ile195 200 205Glu Lys Ala Glu Ser Thr Lys Glu Val Arg Lys Lys Asn Val Arg Ser210 215 220Lys Lys Glu Glu Ser Glu Ile Pro Lys Asn Ala Leu Thr Ile Leu Lys225 230 235 240Pro Gln Thr Gly Lys Lys Leu Glu Ser Lys Ser Leu Phe Ser Ser Leu245 250 255Thr Asn Gln Asn Ser Thr Glu Glu Phe Ser Ser Asn Pro Gln Phe Arg260 265 270Glu Ile Glu Ser Asn Val Ile Arg Phe Glu Lys Asn Ser Ile Gln Ile275 280 285His Lys Glu Ser Arg Pro Ser Leu Asn Arg Leu Ala Arg Trp Met Lys290 295 300Gln Asp Ser Ser Ile Arg Val Lys Val Ile Gly His Thr Ser Leu Glu305 310 315 320Gly Ser Glu Asp Ala Asn Gln Lys Val Ser Leu Leu Arg Ala Gln Thr325 330 335Val Arg Asn Tyr Ile Ala Gly Asn Gly Ile Ser Lys Asp Arg Phe Glu340 345 350Ile Ile Pro Lys Gly Ala Ser Val Pro Ile Gly Asp Asn Ser Lys Glu
355 360 365Glu Gly Lys Glu Met Asn Arg Arg Val Glu Leu Arg Ile Tyr Asn370 375 380<210>27<211>459<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>27Met Arg Ile Lys Arg Asp His Leu Pro Glu Asn Tyr Lys Ser Ile Phe1 5 10 15Tyr Phe Leu Lys Leu Phe Leu Ile Leu Met Lys Asn Val Ala Asp Asp20 25 30Phe Leu Asn Leu Pro Ile Leu Phe Asn Thr Ile Phe Phe Leu Ala Ile35 40 45Leu Val Cys Pro Val Arg Ser Glu Asn Ser Ser Gln Leu Asp Val Arg50 55 60Thr Ile Leu Asp Lys Ala Glu Lys Asn Ser Pro Leu Leu Leu Ser Leu65 70 75 80Asn Ala Asp Leu Glu Ser Leu Phe Tyr Gln Arg Lys Gln Gln Gly Lys85 90 95Thr Gln Asn Pro Ser Leu Thr Leu Asp Tyr Gly Arg Arg Ser Ala Ala100 105 110Asn Glu Ser Gly Ser Glu Tyr Ala Leu Gln Phe Glu Gln Pro Val Tyr115 120 125Phe Pro Gly Arg Lys Glu Leu His Gln Leu Leu Val Asp Asn Asp Ser130 135 140Lys Ile Lys Glu Ile Gln Leu Gln Glu Ala Asn Asn Ser Ile Arg Phe145 150 155 160Asn Ala Leu Lys Phe Ala Tyr Arg Tyr Leu Val Ser Ala Gly Lys Lys165 170 175Asn His Val Lys Glu Arg Leu Lys Arg Leu Ser Ile Leu Glu Ser Tyr180 185 190Ile Arg Ala Arg Pro Phe Ile Thr Pro Gln Ser Lys Thr Asp Leu Phe195 200 205Ile Ile Gln Arg Lys Ile Leu Ala Leu Arg Lys His Phe Asn Asp Leu210 215 220Glu Leu Asp Ala Asn Lys Gln Tyr Glu Ala Met Asn Leu Tyr Leu Met225 230 235 240Leu Glu Ser Val Pro Ser Phe Arg Ile Pro Phe Phe Thr Glu Gly Val245 250 255Lys Phe Asn Phe Asn Glu Leu Leu Asn Lys Ala Val Ser Gln Asn Leu260 265 270Thr Leu Met Ala Ala Lys Gly Glu Ile Glu Lys Ala Lys Thr Glu Leu
275 280 285Asn Leu Ala Ser Leu Glu Lys Tyr Pro Asp Tyr Ser Ile Ile Ser Gln290 295 300Val Gly Glu Asp Arg Ser Gly Val Ala Asn Arg Phe Tyr Asp Phe Gly305 310 315 320Phe Lys Phe Arg Ile Pro Val Trp Asp Arg Phe Gln Asn Lys Ile Ser325 330 335Ala Ala Glu Val Asn Val Lys Ser Lys Gln Glu Ile Phe Arg His Gln340 345 350Glu Asn Leu Val Lys Thr Ala Phe Lys Gln Ala Phe Leu Asp Tyr Glu355 360 365Gln Ser Lys Ile Asn Leu Lys Leu Phe Asn Leu Ser Lys Leu Asp Glu370 375 380Ile Glu Lys Asp Leu Ile Tyr Ala Asp Val Glu Phe Lys Lys Gly Arg385 390 395 400Ile Leu Met Ile Ser Tyr Leu Glu Leu Glu Asn Gln Leu His Glu Thr405 410 415His His Ala Ile Leu Asp Ala Gln Ile Ala His Leu Glu Ala Leu Leu420 425 430Asn Leu Leu Tyr Ile Thr Asn Glu Lys Glu Ile Ile Gly Thr Leu Gln435 440 445His Ala Val Gln Thr Phe Glu Tyr Gln Leu Lys450 455<210>28<211>557<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>28Met Tyr Tyr Lys Lys Lys Arg Thr Leu Glu Lys Phe Leu Val Phe Gly1 5 10 15Thr Ile Leu Leu Thr Met Ile Gln Pro Thr Trp Ser Glu Asp Ile Leu20 25 30Pro Glu Glu Thr Val Leu Glu Glu Asn Lys Lys Ser Pro Val Glu Asn35 40 45Leu Gly Asp Ser Lys Lys Ile Leu Arg Leu Thr Leu Lys Asp Ala Val50 55 60Asn Tyr Val Leu Glu Lys Asn Ile Thr Ile Gln Asn Ala Lys Met Glu65 70 75 80Tyr Val Lys Ala Asp Gly Gly Glu Leu Lys Asn Glu Ser Gln Phe Thr85 90 95Trp Asn Leu Ile Gly Gly Ile Thr Val Phe Arg Thr Thr Leu Pro Ala100 105 110Asn Arg Asn Asn Ile Phe Ala Gly Thr Lys Gln Ser Gln Asp Lys Leu
115 120 125Ser Val Gly Ile Glu Lys Asn Phe Arg Thr Gly Thr Tyr Ala Lys Leu130 135 140Glu Ala Ser Thr Thr Arg Phe Asp Thr Ser Ala Phe Glu Asn Pro Ser145 150 155 160Thr Thr Pro Ser Asn Leu Ala Ala Leu Ala Ile Pro Pro Leu Tyr Thr165 170 175Gly Ala Leu Thr Ile Thr Leu Ser Gln Glu Ile Leu Lys Tyr Ser Phe180 185 190Gly Lys Thr Gln Lys Glu Arg Glu Ala Ile Leu Arg Gln Asn Thr Val195 200 205Ile Lys Arg Glu Glu Leu Ile Tyr Val Leu Ser Gln Leu Val Ala Gln210 215 220Thr Leu Ile Gln Tyr Trp Ser Leu Asn Ile Tyr Asp Ser Asn Val Lys225 230 235 240Thr Leu Gln Asp Leu Glu Ser Asn Thr Arg Asn Ile Arg Asp Leu Thr245 250 255Val Arg Lys Arg Asn Leu Gly Leu Ser Glu Gly Phe Glu Val Asn Leu260 265 270Trp Asn Ser Ile Leu Ser Gln Thr Ala Gly Asn Leu Glu Lys Ala Lys275 280 285Val Ser Arg Lys Glu Ala Glu Arg Asn Leu Ile Arg Ile Leu Asn Ala290 295 300Asp Pro Ser Ser Lys Ile Glu Gly Val Thr Asp Leu Gln Glu Asn Val305 310 315 320Pro Leu Asp Phe Asn Val Glu Lys Asp Tyr Ile Tyr Ala Leu Asp His325 330 335Arg Thr Asp Leu Lys Asn Leu Arg Lys Gln Arg Glu Ile Ala Glu Leu340 345 350Asn Leu Lys Ile Lys Glu Ala Glu Asp Met Pro Ser Leu Lys Leu Ser355 360 365Gly Ala Tyr Ser Thr Arg Gly Gln Asn Ile Val Ser Pro Gln Gln Asn370 375 380Leu Thr Asp Gly Asn Arg Gly Val Ala Ser Phe Lys Tyr Pro Glu Ala385 390 395 400Tyr Ala Ala Phe Gln Phe Ser Tyr Pro Leu Trp Asp Lys Gly Ile Lys405 410 415Ala Asp Ile Arg Asn Ala Lys Leu Asp Val Gln Asn Leu Glu Lys Lys420 425 430Glu Ala Glu Leu Lys Leu Ser Ile Lys Glu Glu Leu Glu Asn Arg Tyr435 440 445Ala Ala Ile Val Ala Asp Lys Asp Ile Phe Glu Gly Ala Lys Lys Arg450 455 460Lys Glu Glu Ala Asn Lys Phe Tyr Lys Gly Leu Ser Glu Arg Phe Arg465 470 475 480Gln Gly Arg Phe Thr Ala Val Ala Val Lys Asn Ala Leu Asp Asn Val485 490 495Ile Gln Ser Glu Leu Gln Val Thr Gln Ala Lys Ile Gln Leu Asn Ile500 505 510Asp Ile Leu Arg Tyr Glu Leu Ala Lys Asn His Ile Phe Glu Arg Phe515 520 525Gly Val Asn Val Asn Asp Ile Ile Asp Arg Leu Met Lys Met Val Asp530 535 540Ile Ala Gln Ser Lys Ser Ser Thr Glu Thr Ser Glu Lys545 550 555<210>29<211>533<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>29Met Arg Glu Ser Lys Glu Ile Gln Met Glu Asn Lys Phe Lys Asn Trp1 5 10 15Lys Gln Arg Ile Leu Lys Phe Gln Arg Leu Ser Leu Phe Leu Leu Gly20 25 30Asn Leu Ile Leu Leu Ser Ser Thr Leu Phe Ala Glu Ser Ser Thr Leu35 40 45Lys Leu Thr Thr Glu Glu Thr Val Lys Arg Ala Leu Glu Ser Asn Tyr50 55 60Asn Leu Gln Asn Leu Arg Tyr Glu Leu Ala Lys Ser Asp Thr Asn Phe65 70 75 80Leu Lys Asn Asp Ser Lys Tyr Ser Trp Arg Leu Val Ala Asp Gly Arg85 90 95Ser Ser Gln Ser Ile Leu Pro Phe Asn Gln Ala Asn Phe Phe Thr Gly100 105 110Thr Lys Ile Ser Asp Asp Thr Ile Lys Gly Gly Ile Glu Lys Val Leu115 120 125Gln Thr Thr Gly Thr Tyr Phe Lys Ile Glu Ala Gly Ser Arg Arg Phe130 135 140Asp Ser Asn Ala Phe Glu Asn Pro Ser Thr Thr Pro Ala Gly Phe Ser145 150 155 160Ser Leu Gly Ile Pro Pro Leu Tyr Thr Gly Phe Val Arg Ala Thr Ile165 170 175Ser Gln Asp Leu Leu Lys Asn Ser Phe Gly Tyr Lys Gly Arg Asn Glu180 185 190Val Lys Ile Leu Glu Ser Gln Ala Glu Ile Val Lys Asn Gln Val Ser195 200 205Gln Gln Ile Ser Ser Val Ile Val Glu Ser Leu Val Asp Phe Trp Asp210 215 220Tyr Ser Ile Lys Thr Gln Ala Val Lys Thr Tyr Lys Gln Leu Val Glu225 230 235 240Asn Thr Arg Asn Ile Arg Asn Leu Thr Ala Arg Lys Gln Ser Leu Gly245 250 255Leu Ser Glu Ser Phe Glu Val Asn Gln Trp Asn Ala Leu Leu Ala Gln260 265 270Ala Gln Asn Gln Leu Glu Thr Ala Gln Val Gln Lys Glu Glu Ser Lys275 280 285Arg Lys Leu Ile Arg Ser Leu Lys Ile Pro Asp Asp Thr Thr Leu Ser290 295 300Glu Glu Thr Asn Leu Leu Glu Glu Leu Thr Glu Lys Pro Glu Tyr Thr305 310 315 320Lys Asp Leu Glu Tyr Ala Tyr Lys His Arg Ala Asp Phe Leu Asn Ala325 330 335Leu Lys Gln Lys Glu Ile Ala Glu Ala Ala Leu Lys Asn Ala Asn Asn340 345 350Asp Arg Leu Pro Thr Leu Thr Leu Ser Gly Thr Gly Ala Ser Gln Ala355 360 365Gln Asn Ile Ile Ser Pro Gln Asp Asn Tyr Asn Asp Thr Asn Gln Gly370 375 380Ile Thr Thr Ala Lys Tyr Lys Glu Trp Thr Gly Gln Val Asn Phe Val385 390 395 400Tyr Pro Ile Ala Asp Lys Gly Ile Tyr Ala Gly Val Arg Asp Ala Asn405 410 415Ile Gly Met Arg Gln Ala Thr Leu Lys Glu Glu Glu Leu Lys Asn Glu420 425 430Val Arg Asp Asp Val Lys Thr Arg Ile Glu Ala Leu Glu Ala Ser His435 440 445Arg Ile Tyr Lys Asn Asn Ile Val Thr Glu Arg Glu Thr Ala Asn Tyr450 455 460Tyr Asn Gly Val Leu Arg Ser Phe Arg Gln Gly Arg Ala Asp Ala Val465 470 475 480Ala Val Lys Asn Ala Leu Asp Thr His Val Gln Asp Gln Leu Arg Leu485 490 495Thr Gln Ala Lys Val Asn Phe Asn Ile Asp Leu Leu Arg Tyr Tyr Leu500 505 510Ala Lys Asn Ala Leu Met Glu Arg Phe Gln Val Asp Arg Asp Lys Leu515 520 525Ile Pro His Leu Asp530<210>30<211>344<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>30Met Lys Thr Leu Gln Gln Lys Ile Thr Phe Pro Ile Leu Val Gly Leu1 5 10 15Ile Ser Val Thr Leu Ile Met Leu Ile Trp Phe Val Phe Phe Ser Gly20 25 30Ser Lys Ile Thr Thr Ser Ser Ser Ala Ser Leu Asp Pro Glu Ser Ser35 40 45Ser Gly Ser Glu Gly Trp Ile Leu Asn Gln Ala Ile Ile Asn Thr Ser50 55 60Arg Lys Ile Phe Asp Glu Asn Gly Asn Trp Leu Ser Phe Asp Glu Leu65 70 75 80Ile Gln Tyr Ala Ser Asn Gly Glu Ile Asn Leu Ile Ser Glu Leu Ser85 90 95Asp Leu Arg Arg Gln Cys Pro Glu Asn Ile His Tyr Glu Gln Cys Asn100 105 110Glu Ile Ile Arg Ala Phe Ile Ala Asp His Tyr Phe Gly Lys Asp Ala115 120 125Glu Tyr Leu Met Lys Leu Phe Ser Ser Tyr Leu Lys Tyr Glu Thr Lys130 135 140Met Lys Glu Leu Glu Ile Ser Asp Lys Leu Ser Arg Ala Glu Lys Tyr145 150 155 160Glu Leu Ile Lys Lys Gln Arg Arg Glu Phe Phe Ser Asp Lys Asp Thr165 170 175Lys Leu Ile Phe Gly Leu Glu Glu Ala Glu Glu Thr Tyr Leu Asp Ser180 185 190Leu Gly Gly Phe Leu Lys Asp Thr Glu Thr Leu Ser Gly Glu Gln Arg195 200 205Met Gln Lys Tyr Glu Glu Phe Arg Lys Asn Val Tyr Gly Gln Tyr Tyr210 215 220Asn Thr Ile Lys Lys Arg Glu Pro Lys Tyr Asn Thr Tyr Glu Thr Glu225 230 235 240Met Phe Leu Arg Glu Lys Glu Leu Glu Arg Met Ser Ser Ser Glu Arg245 250 255Asn Ser Lys Thr Arg His Ile Arg Glu Lys Tyr Phe Gly Lys Asp Gly260 265 270Ala Asp Arg Met Glu Thr Val Tyr Gln Glu Ser Glu Glu Lys Glu Lys275 280 285Lys Glu Lys Gln Thr Ala Gln Glu Glu Ser Asp Trp Ile Arg Lys Asn290 295 300Pro Asn Val Lys Val Glu Thr Lys Glu Lys Ala Leu Met Glu Ile Arg305 310 315 320Ile Lys Asn Leu Gly Lys Glu Glu Ala Glu Glu Tyr Ser Arg Arg Leu325 330 335Lys Tyr Glu Glu Glu Ile Lys Lys
340<210>31<211>159<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>31Met Lys Lys His Ser Ile Ser Lys Ile Ile Ile Thr Ala Cys Cys Ile1 5 10 15Phe Leu Leu Thr Tyr Gly Cys Lys Gln Asp Pro Val Asp Tyr Asn Asn20 25 30Lys Ile Met Glu Ile Met Asn Ala Ser Thr Asn Asp Leu Asp Ala Leu35 40 45Asn Ala Ala Met Glu Lys Glu Asp Leu Thr Asn Ala Glu Asn Val Arg50 55 60Lys Ala Trp Glu Thr Lys Leu Val Ser Ser Leu Asp Lys Leu Lys Gly65 70 75 80Ile Ser Asp Phe Lys Gly Asp Ser Ser Phe Lys Asn Ala Ser Val Gln85 90 95Ala Leu Glu Thr Tyr Leu Asn Ile Val Ser Lys Asp Tyr Lys Arg Leu100 105 110Ile Glu Leu Arg Gly Leu Gly Asp Lys Ala Asp Ser Asn Glu Ile Asn115 120 125Gln Val Leu Asn Arg Ile Asn Gln Asp Phe Glu Lys Ala Ala Asn Thr130 135 140Leu Asn Ala Ala Ser Asp Lys Phe Ala Lys Glu Tyr Ala Ser Gln145 150 155<210>32<211>551<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>32Met Met Arg Ile Lys Thr Ile Asn Leu Val Ala Ser Val Phe Cys Leu1 5 10 15Leu Val Ser Ser Gln Leu Leu Ser Gln Thr Gly Lys Lys Pro Ala Ala20 25 30Asp Thr Val Ala Pro Ala Pro Pro Ser Gln Thr Ile Glu Leu Glu Glu35 40 45Lys Lys Trp Tyr Asp Gln Val Glu Phe Ser Gly Phe Ala Asp Val Tyr50 55 60Tyr Met Tyr Asn Leu Asn Pro Lys Gln Gly Asn Asp Ile Asp Ser Thr65 70 75 80Arg Ala Phe Glu Thr Ser Asn Lys Asn Phe Ala Val Asn Ala Val Ala
85 90 95Leu Thr Ile Gln Lys Ala Ala Glu Lys Ser Ser Pro Trp Gly Phe Arg100 105 110Ile Asp Ile Gln Asn Gly Gln Asn Asn Ala Phe Gln Glu Ala Pro Tyr115 120 125Ser Gln Ser Asn Ser Ile Tyr Asn Tyr Asn Met Leu Lys Gln Ala Tyr130 135 140Val Ser Leu Tyr Phe Pro Val Leu Lys Gly Met Thr Leu Asp Val Gly145 150 155 160Lys Met Ala Thr His Ile Gly Tyr Glu Val Leu Glu Ser Met Ser Asn165 170 175Pro Asn Tyr Ser Ile Gly Ala Ile Phe Gln Asn Thr Ile Pro Phe Ile180 185 190His Thr Gly Ala Arg Leu Thr Thr Gln Phe Thr Asp Lys Trp Ala Gly195 200 205Thr Phe Tyr Val Tyr Asn Ser Gly Ala Gly Thr Gly Tyr Asn Ser Pro210 215 220Ala Thr Val Thr Thr Ala Pro Phe Asn Val Ile Thr Asp Pro Thr Tyr225 230 235 240Ser Gly Asn Ala Ala Gly Lys Ser Tyr Phe Val Glu Gly Gln Thr Glu245 250 255Arg Lys Ala Ile Gly Thr Gln Ile Lys Gly Gln Leu Ile Glu Asp Lys260 265 270Leu Ser Val Thr Trp Asn Thr Leu Tyr Ser Ser Asp Gly Ala Tyr Ala275 280 285Arg Val Asp Pro Ser Lys Ala Ala Met Ala Thr Glu Leu Ser Ser Val290 295 300Leu Gly Asp Pro Asn Ile Ala Lys Tyr Asn Val Thr Asn Pro Ala Arg305 310 315 320Ala Lys Tyr Asn Lys Asp Tyr Trp Phe Met Asn His Ala Ile Leu Ser325 330 335Ile Thr Pro Thr Asp Lys Ile Thr Ile Asp Leu Asp Tyr Thr Trp Ser340 345 350Glu Lys Ser Gly Ala Leu Ala Asn Asn Ser Leu Asp Gln Arg Arg Tyr355 360 365Asn Val Asp Thr Asn Thr Ala Glu Val Phe Asn Lys Asp Thr Leu Phe370 375 380Trp Gly Leu Asn Asn Lys Arg Asp Leu Lys Thr Thr Tyr Lys Ala Tyr385 390 395 400Gly Ile Phe Ala Lys Phe Lys Ile Asn Glu Asn Trp Gly Val Asn Val405 4l0 415Arg Ala Glu Tyr Ile Asp Asp Lys His Asn Asn Gly Ala Leu Thr Ile420 425 430Phe Asn Pro Phe Met Gly Pro Asn Thr Tyr Leu Ser Glu Phe Lys Lys435 440 445Leu Thr Asp Ala Ala Ile Ala Asp Ala Val Val Ala Ser Leu Ala Ala450 455 460Asp Pro Asn Leu Gly Pro Leu Gly Ile Thr Lys Glu Gln Ile Leu Glu465 470 475 480Val Met Ser Asp Asp Tyr Lys Asn Tyr Gly Gly Thr Arg Asn Ala Gly485 490 495Gln Tyr Lys Thr Phe Thr Val Thr Pro Val Trp Asn Phe Thr Glu Asn500 505 510Leu Leu Ile Lys Leu Asp Leu Arg Arg Asp Trp Ala Thr Gly Lys Gln515 520 525Phe Val Asp Gln Lys Gly Asp Lys Thr Asp His Gln Tyr Gly Met Thr530 535 540Leu Gly Ile Val Ala Lys Phe545 550<210>33<211>276<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>33Met Lys Leu Gln Lys Leu Phe Leu Ala Val Leu Ile Ala Ile Ser Thr1 5 10 15Ala Val Phe Ser Gln Gln Asn Ser Gly Ser Asp Gln Lys Ser Gln Pro20 25 30Ser Ser Ala Gln Leu Gly Gln Ser Ile Leu Glu Thr Glu Arg Lys Leu35 40 45Asp Glu Lys Ile Phe Glu Leu Asn Gln Arg Leu Thr Arg His Thr Val50 55 60Leu Met Lys Met Lys Val Arg Val Leu Pro Phe Arg Thr Val Leu Phe65 70 75 80Lys Gly Lys Ala Asn Asn Asp Glu Cys Thr Pro Ala Ile Asn Gln Glu85 90 95Asp Pro Ala Asn Asn Cys Ile Arg Val Glu Val Tyr Asp Phe Ile Arg100 105 110Asp Glu Glu Arg Gly Leu Asn Lys Asn Val Gln Gly Ala Leu Ala Lys115 120 125Tyr Met Glu Ile Tyr Phe Glu Gly Gln Asn Ser Asn Asp Pro Glu Pro130 135 140Arg Thr Glu Pro Pro Arg Asn Ile Asn Lys Leu Lys Ser Lys Ile Tyr145 150 155 160Lys Asn Asn Met Val Leu Glu Asp Lys Ile Ile Ser Glu Val Met Asp165 170 175Arg Gly Pro Asn Thr Gln Pro Ser His Asn Asp Lys Val Glu Val Phe180 185 190Phe Gln Lys Asp Asn Tyr Pro Glu Tyr Gly Arg Pro Glu Thr Pro Ala195 200 205Glu Lys Gly Val Gly Lys Tyr Ile Leu Ala Gly Val Glu Asn Thr Lys210 215 220Thr His Pro Ile Arg Asn Ser Phe Lys Lys Glu Phe Tyr Ile Lys His225 230 235 240Leu Asp Gln Phe Asp Arg Leu Phe Thr Lys Ile Phe Asp Tyr Asn Asp245 250 255Gln Leu Gly Asn Glu Asn Tyr Lys Glu Asn Val Asp Ala Leu Lys Asp260 265 270Ser Leu Arg Tyr275<210>34<211>662<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>34Met Asn Gln Ala Asn Ile Lys Ala Gly Phe His Phe Val Phe Cys Ile1 5 10 15Phe Leu Ile Phe Gly Ile Cys Phe Phe Pro Ile Ser Val Phe Ser Gln20 25 30Ser Glu Cys Gly Asn Ser Pro Pro Glu Tyr Phe Leu Ser Leu Ala Phe35 40 45Glu Arg Gln Ala Asn Ala Ala Arg Ile Gln Ser Gln Glu Gln Phe Gln50 55 60Glu Glu Ser Lys Lys Ile Tyr Glu Glu Ser Leu Glu Tyr Tyr Asn Arg65 70 75 80Tyr Ile His Cys Ser Glu Glu Leu Gln Lys Glu Val Ser Pro Val Ser85 90 95Arg Val Ala Lys Ala Ser Val His Phe Phe Leu Glu Gln Leu Glu Asn100 105 110Ala Ser Lys Glu Val Asp Ala Ala Leu Ile Ser Asp Ser Ser Phe Arg115 120 125Asp Gly Tyr Ile Leu Lys Ala Lys Ile Leu Ile Arg Phe Gly Glu Phe130 135 140Gln Lys Ala Ser Asp Phe Leu Glu Thr Asn Leu Ser Arg Phe Pro Asp145 150 155 160Asp Ser Asp Phe Leu Tyr Leu Leu Gly Thr Leu Asn Gln Glu Leu Lys165 170 175Asn Tyr Pro Arg Ala Ile Leu Tyr Leu Thr Ser Leu Ser Asp Thr Ile180 185 190Arg Asn Arg Glu Gly Asn Pro Lys Tyr Arg Ser Phe Val Asp Lys Ser195 200 205Leu Gly Glu Ile Tyr Phe Ala Asn Ala Gln Pro Lys Lys Ala Leu Tyr210 215 220Phe Leu Asn Ser Tyr Leu His Gln Asn Pro Ser Asp Leu Ser Ala Arg225 230 235 240Leu Thr Leu Ala Lys Ile Trp Asn Gln Leu Gly Lys Phe Ser Ser Ala245 250 255Arg Lys Glu Leu Asn Arg Ile Leu Lys Thr Lys Lys Asn Leu Ser Ser260 265 270Val Glu His Leu Leu Ala Glu Met Tyr Phe Ile Glu Ser Arg Ala Ser275 280 285Ala Phe Glu Tyr Phe Asn Ile Leu Asn Lys Asn Ser Lys Ile Pro Lys290 295 300Gly Gly Val Leu Glu Gly Leu Tyr Leu Ile Leu Gln Gly Arg Thr Lys305 310 315 320Gln Ala Lys Val Leu Leu Leu Pro Val Lys Glu Lys Phe Pro Ser Arg325 330 335Leu Ala Val Arg Leu Ala Ile Leu Asp Val Tyr Glu Thr Glu Asn Asn340 345 350Thr Ser Leu Phe Leu Lys Glu Leu Lys Glu Val Ala Glu Leu Val Phe355 360 365Gly Met Gln Gln Phe Glu Leu Ala Glu Arg Ile Ala Gln Arg Ala Leu370 375 380Ser Val Ser Asn Lys Ser Ser Glu Trp Ser Lys Ser Glu Ile Tyr Asp385 390 395 400Phe Leu Ala Ser Cys His Glu Gln Ser Gly Tyr Ile Tyr Arg Ala Ile405 410 415Leu Met Ser Arg Lys Ala Val Glu Asn Ser Lys Lys Glu Glu Glu Thr420 425 430Leu Lys Phe Gln Leu His Leu Ala Tyr Leu Leu Arg Asn Asp Pro Pro435 440 445Asp Lys Lys Glu Glu Ala Glu Thr Leu Ile Arg Asn Val Leu Gln Ile450 455 460Arg Pro Glu Met Ser Tyr Ala Arg Tyr Leu Leu Gly Val Val Leu Ala465 470 475 480Ser Asn Glu Lys Tyr Lys Glu Ala Leu Glu Glu Leu Asn Val Ala Ile485 490 495Glu Thr Asp Pro Glu Asn Pro Val Tyr Tyr Phe Tyr Arg Ala Ser Val500 505 510His Glu Arg Leu Glu Gln Gln Glu Phe Met Glu Arg Asp Leu Lys Lys515 520 525Ser Ile Glu Ile Asp Pro Gly Asn Pro Val Ala Tyr Asn Tyr Leu Gly530 535 540Tyr Tyr Leu Ser Glu Lys Gly Val Arg Leu Glu Glu Ser Leu Ser Leu545 550 555 560Val Gln Lys Ala Val Glu Leu Ala Pro Asp Asn Glu Ala Tyr Gln Asp
565 570 575Ser Leu Gly Trp Ile Phe Phe Lys Leu Gly Asn His Glu Glu Ala Leu580 585 590Leu His Leu Gln Leu Ala Tyr Gln Ile Leu Lys Asp Lys Gly Gly Glu595 600 605Asp Pro Val Ile Leu Glu His Leu Gly Asp Val Tyr Lys Glu Lys Asn610 615 620Gln Gly Gly Asn Ala Val Ala Tyr Trp Glu Lys Ser Leu Lys Leu Phe625 630 635 640Lys Lys Lys Glu Asp Ile Tyr Arg Ile Gln Lys Lys Ile Gln Thr Gly645 650 655Val Arg Gln Asn Asn Lys660<210>35<211>519<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>35Met Asn Glu Gln Ala Glu Ser Lys Ser Glu Thr Ala Lys Leu Val Aspl 5 l0 15Lys Lys Arg Ile Ala Ile Cys Gly Gly Thr Leu Gly Arg Glu Asn Arg20 25 30Tyr Tyr Val Arg Gly Gln Val Val Asp Val Gly Ile Thr Glu Glu Met35 40 45Arg Asp Asp Ser Arg Trp Asp Leu Leu Asn Gly Leu Phe Glu Gly Gln50 55 60Glu Lys Glu Ile Thr Pro Phe Leu Asp Tyr Gly Leu Glu Pro Val Arg65 70 75 80Lys Pro Ile Leu Val Ala Glu Ile Trp Asp Glu Ser Asp Val Leu Val85 90 95His Gln Ser Pro Glu Ile Lys Gly Asp Glu Gly Gly Phe Phe Phe His100 105 110Glu Phe Thr Lys Pro Leu Pro Pro Gly Lys Tyr Ile Phe Gln Ile Arg115 120 125Phe Arg Lys Leu Asp Ser Tyr Arg Gln Phe Thr Lys Asp Ile Ala Tyr130 135 140Leu Asn Gln Lys Gly Lys Ser Glu Ile Ser Gly Gln Ser Leu Ile Gly145 150 155 160Lys Gly Lys Leu Arg Ile Leu Pro Glu Asn Phe Asn Gly Tyr Val Thr165 170 175Thr Ser Asp Ile Asp Gln Thr Tyr Leu Ala Thr Asp Ile His Ser Asn180 185 190Lys Gly Lys Leu Ser Thr Leu Phe Glu Thr Pro Gln Gln Lys Leu Pro
195 200 205Leu Pro Gly Met Pro Ala Leu Tyr Arg Glu Ile Arg Leu Ala Thr Glu210 215 220Asp Ser Pro Leu Cys Phe Ile Ser Ala Ser Pro His Phe Phe Arg Arg225 230 235 240Thr Leu Leu Ser Thr Ile Gln Ser His Ser Ile Thr Thr Glu Ser Leu245 250 255His Leu Lys Tyr Leu Glu Gly Thr Ile Lys Gly Ile Val Glu Lys Phe260 265 270Trp Asp Ser Val Thr His Pro Ala Lys Phe Ile Thr Asp Gly Ile Leu275 280 285Gly Ser Met Glu Arg Ile Arg Lys Phe Ala Gly Ala Ser Phe Gln Ser290 295 300Leu Phe Asp Gln Met Ser Tyr Lys Leu Thr Ile Leu Leu Arg Asp Arg305 310 315 320Leu Tyr Leu Pro Thr Asn Ala Lys Glu Ile Leu Ile Gly Asp Asn Thr325 330 335Glu Ser Asp Tyr Leu Ile Phe Ile Leu Tyr Gln Tyr Ile Leu Cys Gly340 345 350Lys Met Gln Gly Lys Glu Leu Glu Asp Tyr Leu Tyr Arg Leu Asn Phe355 360 365Leu Gly Arg Asp Ala Ile Thr Arg Asp Ala Ala Arg Ile Ile Arg Glu370 375 380Leu Gly Glu Glu Asn Arg Asn Ile His Gly Asp Leu Asn Ser Val Ser385 390 395 400Leu Val Leu Ile Asn Lys Thr Val His Gly Pro Asp Gln Glu Glu Met405 410 415His Trp Asn Ile Gln Ser Ala Leu Pro Ala Gly Ile Asp Pro Phe Lys420 425 430Gln Lys Glu Ile His Pro Tyr Ile Leu Thr Glu Gly Ala Pro Gly Phe435 440 445Ala Val Val Leu Gln Asp Asn Gly Ile Leu Asp Thr Ser Ala Val Phe450 455 460Arg Ile Val Ala Glu Met Ala Gly Glu Trp Met Glu Gly Lys Val Ile465 470 475 480Asp Ser Thr Ala Leu Met Glu Trp Ile Gln Asn Leu Thr Leu Pro Gly485 490 495Asp Tyr Gln Asn Glu Lys Asp Leu Ile Leu Glu Gly Leu Lys Lys Ala500 505 510Leu Lys Lys Glu Glu Ile Ser515<210>36<211>167<212>PRT<213>鉤端螺旋體(Leptospira interrogans)<400>36Met Val Gln Arg Gly Leu Ile Arg Asp Val Phe Asp Glu Asn Thr Val1 5 10 15Pro Asp Asn Gly Gly Asp Gln Ile Glu Lys Asn His Ile Leu Gly Val20 25 30Lys Thr Asn Tyr Thr Val Arg Gly Phe Asp Arg Val Glu Val Phe Pro35 40 45Pro His Glu Tyr Val Val Lys Gly Lys Ala Arg Gln Leu Ser Ile Trp50 55 60Ala Leu Gly Arg Lys Phe Arg His Thr Leu Phe Ala Lys Leu Arg Asp65 70 75 80Tyr Arg Gly Asn Thr His Asn Ile Arg Leu Gly Arg Leu Asp Tyr Phe85 90 95Gly Trp Arg Lys Leu Thr Ala Thr Ile Pro Gly Phe Val Pro Gln Ser100 105 110Thr Arg Phe Ala Leu Leu Asp Lys Asn Leu His Phe Val Ser Leu Phe115 120 125Val Val Ser Asp Val His Glu Val Gly Gly Gln Phe Tyr Phe Tyr Val130 135 140Asp Asp Leu Glu Val Arg Ala Asp Lys Ser Asp Ala Lys Tyr Pro Gly145 150 155 160Ser Glu Ile Lys Asp Asn Trp16權(quán)利要求
1.一種分離的鉤端螺旋體外膜蛋白多肽，其特征在于，它包含具有選自SEQ ID NO2-26中的偶數(shù)序列和SEQ ID NO27-36的氨基酸序列的多肽、或其保守性變異多肽、或其活性片段、或其活性衍生物。
2.如權(quán)利要求1所述的多肽，其特征在于，該多肽是具有SEQ ID NO2-26中的偶數(shù)序列和SEQ ID NO27-36的氨基酸序列的多肽。
3.一種分離的多核苷酸，其特征在于，它包含一核苷酸序列，該核苷酸序列與選自下組的一種核苷酸序列有至少70％相同性(a)編碼如權(quán)利要求1和2所述多肽的多核苷酸；(b)與多核苷酸(a)互補(bǔ)的多核苷酸。
4.如權(quán)利要求3所述的多核苷酸，其特征在于，該多核苷酸編碼具有SEQ IDNO2-26中的偶數(shù)序列和SEQ ID NO27-36所示氨基酸序列的多肽。
5.如權(quán)利要求3所述的多核苷酸，其特征在于，該多核苷酸的序列選自下組(a)具有SEQ ID NO1-26中的奇數(shù)序列。
6.一種載體，其特征在于，它含有權(quán)利要求3所述的多核苷酸。
7.一種遺傳工程化的宿主細(xì)胞，其特征在于，它含有權(quán)利要求6所述的載體。
8.一種鉤端螺旋體OMP蛋白多肽的制備方法，其特征在于，該方法包含(a)在適合表達(dá)鉤端螺旋體OMP蛋白的條件下，培養(yǎng)權(quán)利要求7所述的宿主細(xì)胞；(b)從培養(yǎng)物中分離出鉤端螺旋體OMP蛋白多肽。
9.一種能與權(quán)利要求1所述的鉤端螺旋體OMP蛋白特異性結(jié)合的抗體。
10.一種疫苗組合物，其特征在于，它含有安全有效量的權(quán)利要求1所述的多肽和藥學(xué)上可接受的載體。
全文摘要
本發(fā)明提供了一類新的鉤端螺旋體外膜蛋白(OMP蛋白)，編碼OMP蛋白的多核苷酸和經(jīng)重組技術(shù)產(chǎn)生這類OMP蛋白的方法。OMP蛋白是一種有用的制備疫苗的免疫原。本發(fā)明還公開(kāi)了這類OMP蛋白及其編碼序列用于制備疫苗的用途。
文檔編號(hào)C07K14/435GK1443775SQ02110998
公開(kāi)日2003年9月24日 申請(qǐng)日期2002年3月11日 優(yōu)先權(quán)日2002年3月11日
發(fā)明者任雙喜, 傅剛, 曾嶸, 蔣秀高, 張怡軒, 趙國(guó)屏, 陳竺, 繆有剛, 徐海, 郭小奎 申請(qǐng)人:中國(guó)科學(xué)院上海生物工程研究中心