專利名稱：Hla－結(jié)合肽，其前體，編碼這些肽序列的dna片段和重組載體的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及HLA-結(jié)合肽，其前體，編碼這些肽序列的DNA片段和重組載體。
背景技術(shù)：
當(dāng)感染病毒，諸如丙型肝炎病毒(HCV)時，在天生免疫系統(tǒng)防衛(wèi)之后，就會誘導(dǎo)病毒特異的免疫反應(yīng)以消除所述病毒。
當(dāng)誘導(dǎo)特異性免疫反應(yīng)時，分離的病毒粒子通過中和抗體喪失其傳染性，并隨后被消除。換言之，病毒感染的細(xì)胞由細(xì)胞毒性T淋巴細(xì)胞(CTLs)裂解。CTL將HLA I型分子呈遞的表位肽識別成抗原。這種表位肽長度上有8-11個氨基酸。因此，為了開發(fā)針對病毒的治療性疫苗，至關(guān)重要的是鑒別病毒表位肽。
這種技術(shù)從專利公報1是公知的。專利公報1描述了形成自特異性氨基酸序列的寡肽具有與HLA結(jié)合的性質(zhì)。
日本專利申請公開No.H8-151396(1996)發(fā)明內(nèi)容然而，上述專利公報中所述的常規(guī)技術(shù)在下述幾點尚有改進(jìn)的余地。
首先，不清楚上述公報中的HLA-結(jié)合肽是否與HLA分子有效結(jié)合，因此仍有改進(jìn)與HLA結(jié)合的性質(zhì)的余地。
其次，上述公報敘述了其中的HLA-結(jié)合肽具有與HLA-DQ4結(jié)合的性質(zhì)。然而，尚不清楚該肽是否與HLA-A2型分子(HLA-A*0201基因產(chǎn)物等)結(jié)合，該分子在歐美人中是常見的，以及是否與HLA-A24型分子(HLA-A*2402基因產(chǎn)物等)結(jié)合，該分子在日本人是常見的。
在上述情形下，實現(xiàn)了本發(fā)明，并且提供對特異類型的HLA分子顯示高親和性結(jié)合的HLA-結(jié)合肽。
根據(jù)本發(fā)明，提供了與HLA-A型分子結(jié)合的HLA-結(jié)合肽，其含有至少一種氨基酸序列并且由不少于8個且不多于11個氨基酸殘基組成，所述氨基酸序列選自SEQ ID NOS1-183。
而且，根據(jù)本發(fā)明，提供了含有至少一種氨基酸序列的HLA-結(jié)合肽，所述氨基酸序列選自SEQ ID NOS1，2，3，5，8，12，13，14，16，17，18，19，22，23，25，27，34，37，38，40，42，45，48，49，52，53，54，55，56，58，59，60，61，62，63，64，65，67，71，72，74，75，76，84，86，87，90，91，92，93，94，96，97，98，100，101，102，104，106，107，108，109，110，112，123，124，126，127，131，132，133，134，135，136，137，139，141，142，146，147，149，150，152，162，170，173，176，177，和179。
此外，根據(jù)本發(fā)明，提供了與HLA-A型分子結(jié)合的HLA-結(jié)合肽，其含有一種氨基酸序列并且由不少于8個且不多于11個氨基酸殘基，所述氨基酸序列是在上述HLA-結(jié)合肽中所含的氨基酸序列基礎(chǔ)上，通過缺失，替換，或添加一個或兩個氨基酸殘基而形成的。
以此，含有一種氨基酸序列的構(gòu)建體也可顯示與上述HLA-結(jié)合肽相似的效果，所述氨基酸序列是在具有與HLA-A型分子結(jié)合的性質(zhì)的特異氨基酸序列中，通過缺失，替換，或添加一個或數(shù)個氨基酸殘基而形成的。
而且，根據(jù)本發(fā)明，提供了含有為上述HLA-結(jié)合肽編碼的DNA序列的DNA區(qū)段。
此外，根據(jù)本發(fā)明，提供了含有為上述HLA-結(jié)合肽編碼的DNA序列的重組載體。
而且，根據(jù)本發(fā)明，提供了在哺乳動物體內(nèi)變成上述HLA-結(jié)合肽的HLA-結(jié)合肽前體。
根據(jù)本發(fā)明，由于包括特異氨基酸序列，可獲得在與HLA-A型分子結(jié)合方面具有優(yōu)異性質(zhì)的HLA-結(jié)合肽。
附圖簡述上述目的，其他目的，特征和優(yōu)點從下文所述的優(yōu)選實施方案，參照附圖可變得更加顯而易見。
示意圖用于說明實施方案中所用的積極學(xué)習(xí)實驗設(shè)計。
實施本發(fā)明的最佳方式實施本發(fā)明的方式參照附圖解釋如下。在所有附圖中，相同的組成元件以相同的標(biāo)號和符號表示，以便不會重復(fù)解釋。
<實施方案1>
在該實施方案中，含有一種氨基酸序列并由不少于8個且不多于11個氨基酸殘基組成的肽用作HLA-結(jié)合肽的候選物，利用積極學(xué)習(xí)實驗方法(日本專利申請公開No.H11-316754(1999))得到假設(shè)，通過該假設(shè)預(yù)測，所述氨基酸序列與HLA分子結(jié)合的-log Kd值為3或以上。根據(jù)結(jié)合實驗的結(jié)果，已確認(rèn)這些肽真正為HLA-結(jié)合肽。
結(jié)果，可有效獲得大量HLA-結(jié)合肽，由于它們含有一種氨基酸序列，其在與HLA-A型分子結(jié)合方面具有優(yōu)異性質(zhì)，所述氨基酸序列與HLA分子結(jié)合的-log Kd值為3或以上。
具體而言，與該實施方案有關(guān)的HLA-結(jié)合肽為與HLA-A型分子結(jié)合，含有至少一種氨基酸序列(選自SEQ ID NOS1-183，這將在下文描述)，并由不少于8個且不多于11個氨基酸殘基組成的HLA-結(jié)合肽。
在人HLA-A型中，約50％的日本人為HLA-A24型。對于歐美人，諸如德國人，為HLA-A2型。
所有在此所述的序列為由HCV(丙型肝炎病毒)某基因組蛋白中所含的9個氨基酸殘基組成的序列。
序列SEQ ID NOS1-44示于下表1。
表1HLA-A24-結(jié)合肽

序列SEQ ID NOS1-44為由HCV D90208株某基因組蛋白(SEQ IDNO184)中所含的9個氨基酸殘基組成的序列，這將在下文描述。序列SEQ ID NOS1-44為通過上述方法預(yù)測具有優(yōu)良的與HLA-A24型分子結(jié)合的序列。SEQ ID NOS1-44以結(jié)合降低的順序排列。即，SEQ IDNO1為預(yù)測具有最佳結(jié)合的序列。各個序列與HLA-A24型分子結(jié)合的預(yù)測分和結(jié)合實驗數(shù)據(jù)以-log Kd值的形式表示。
序列SEQ ID NOS45-83示于下表2。
表2HLA-A24-結(jié)合肽

序列SEQ ID NOS45-83為由HCV D89815株某基因組蛋白(SEQID NO185)中所含的9個氨基酸殘基組成的序列。序列SEQ IDNOS45-83為通過上述方法預(yù)測具有優(yōu)良的與HLA-A24型分子結(jié)合的序列。SEQ ID NOS45-83以結(jié)合降低的順序排列。即，SEQ ID NO45為預(yù)測具有最佳結(jié)合的序列。各個序列與HLA-A24型分子結(jié)合的預(yù)測分和結(jié)合實驗數(shù)據(jù)以-log Kd值的形式表示。
序列SEQ ID NOS84-123示于下表3。
表3HLA-A24-結(jié)合肽

序列SEQ ID NOS84-123為由HCV pBRT703′X株某基因組蛋白(SEQ ID NO186)中所含的9個氨基酸殘基組成的序列，這將在下文描述。序列SEQ ID NOS84-123為通過上述方法預(yù)測具有優(yōu)良的與HLA-A24型分子結(jié)合的序列。SEQ ID NOS84-123以結(jié)合降低的順序排列。即，SEQ ID NO84為預(yù)測具有最佳結(jié)合的序列。各個序列與HLA-A24型分子結(jié)合的預(yù)測分和結(jié)合實驗數(shù)據(jù)以-log Kd值的形式表示。
序列SEQ ID NOS124-183示于下表4。
表4HLA-A2-結(jié)合肽

序列SEQ ID NOS124-183為由HCV pBRT703′X株某基因組蛋白(SEQ ID NO186)中所含的9個氨基酸殘基組成的序列，這將在下文中描述。序列SEQ ID NOS124-183為通過上述方法預(yù)測具有優(yōu)良的與HLA-A2型分子結(jié)合的序列。SEQ ID NOS124-183以結(jié)合降低的順序排列。即，SEQ ID NO124為預(yù)測具有最佳結(jié)合的序列。各個序列與HLA-A2型分子結(jié)合的預(yù)測分和結(jié)合實驗數(shù)據(jù)以-log Kd值的形式表示。
盡管下文詳細(xì)描述，但在表1-表4中，顯然在預(yù)測分和結(jié)合實驗數(shù)據(jù)之間存在相關(guān)性。即，盡管有小的誤差，但可以說，通過上述方法預(yù)測具有高的與HLA-A型分子結(jié)合的肽經(jīng)實驗發(fā)現(xiàn)具有高的與HLA-A型分子結(jié)合。
既然通過這種實驗設(shè)計方法，沒有用于發(fā)現(xiàn)HLA-結(jié)合肽的常規(guī)技術(shù)，則只有很少量的HLA-結(jié)合肽經(jīng)實驗確認(rèn)具有HLA-結(jié)合性質(zhì)。由此，即使當(dāng)由9個氨基酸殘基組成的肽通過常規(guī)方法隨機合成，進(jìn)行實驗以尋找該肽是否與HLA分子結(jié)合時，則只有約1/100的可能性找到一種結(jié)合的-log Kd值超過6的肽。
根據(jù)該實施方案，如上所述，由于利用通過實驗設(shè)計方法尋找HLA-結(jié)合肽的技術(shù)，可找到多至183個HLA-結(jié)合肽序列。此外，當(dāng)有些獲得的HLA-結(jié)合肽通過實驗檢查結(jié)合時，經(jīng)確認(rèn)，所有已進(jìn)行實驗的序列顯示等于或高于預(yù)測的與HLA的優(yōu)異結(jié)合。
在這些序列中，含有選自下列至少一種氨基酸序列的HLA-結(jié)合肽，通過實驗確認(rèn)與人HLA-A型分子結(jié)合SEQ ID NOS1，2，3，5，8，12，13，14，16，17，18，19，22，23，25，27，34，37，38，40，42，45，48，49，52，53，54，55，56，58，59，60，61，62，63，64，65，67，71，72，74，75，76，84，86，87，90，91，92，93，94，96，97，98，100，101，102，104，106，107，108，109，110，112，123，124，126，127，131，132，133，134，135，136，137，139，141，142，146，147，149，150，152，162，170，173，176，177，和179。因此，可以肯定的說，正是HLA-結(jié)合肽在與人HLA-A型分子結(jié)合方面具有優(yōu)異性質(zhì)。
與本發(fā)明實施方案有關(guān)的HLA-結(jié)合肽與HLA分子結(jié)合的-log Kd值為3或以上，尤其優(yōu)選5或以上，更優(yōu)選5.4或以上。
在生化領(lǐng)域，已知-log Kd值約為3的結(jié)合能力，是肽能否真正與MHC，諸如HLA結(jié)合的閾值。因此，如果與HLA分子結(jié)合的-log Kd值為3或以上，則可以說，其是HLA-結(jié)合肽。
此外，如果與HLA分子結(jié)合的-log Kd值為5或以上，由于所得肽在與HLA分子結(jié)合方面具有優(yōu)異性質(zhì)，則其可合適地用于開發(fā)免疫疾病等的有效治療藥物，預(yù)防藥物等。
而且，如果與HLA分子結(jié)合的-log Kd值為5.4或以上，則所得肽在與HLA分子結(jié)合方面具有尤其良好的性質(zhì)，則其可合適地用于開發(fā)免疫疾病等的更有效治療藥物，預(yù)防藥物等。
此外，可以安排成，與本發(fā)明實施方案有關(guān)的HLA-結(jié)合肽由不少于8個且不多于11個氨基酸殘基組成。
以此方式，如果肽由不少于8個且不多于11個氨基酸殘基組成，則其在與HLA分子結(jié)合方面具有優(yōu)異性質(zhì)。而且，胞毒T淋巴細(xì)胞(CTL)特異性識別病毒抗原(CTL表位)，該抗原由8-11個氨基酸組成，以HLAI型分子被呈遞在感染有病毒等的細(xì)胞的表面上，并通過破壞被感染的細(xì)胞而消除病毒。重要的是制備這種CTL表位，該表位由特異于病毒等的旨在制備針對該病毒等的治療或預(yù)防用疫苗。
例如，上述HLA-結(jié)合肽可以是僅由氨基酸殘基組成的肽，但不特別限于此。例如，它可以是任選修飾以糖鏈或脂肪酸基團(tuán)等的HLA-結(jié)合肽前體，條件是本發(fā)明的效果不被削弱。這種前體發(fā)生變化，包括通過消化酶等在活的哺乳動物體，諸如人消化器官中消化變成HLA-結(jié)合肽，由此顯示與上述HLA-結(jié)合肽所示的效果相似。
此外，上述HLA-結(jié)合肽可以是與人HLA-A24型分子結(jié)合的肽。
而且，上述HLA-結(jié)合肽可以是與人HLA-A2型分子結(jié)合的肽。
根據(jù)該組成，由于獲得的肽與HLA-A24型分子結(jié)合，HLA-A24型分子在亞洲人，諸如日本人中是常見的，因此該肽可用于開發(fā)對亞洲人，諸如日本人尤其有效的治療藥物，預(yù)防藥物等。
此外，根據(jù)該組成，由于獲得的肽與HLA-A2型分子結(jié)合，除日本人之外，HLA-A2型分子還在歐美人中是常見的，因此該肽可用于開發(fā)對除日本人之外還對歐美人尤其有效的治療藥物，預(yù)防藥物等。
上述HLA-結(jié)合肽中所含的氨基酸序列可以是衍生自HCV某基因組蛋白的氨基酸序列，但不特別限于此。例如，其可以是衍生自HCV蛋白的氨基酸序列，衍生自雪松花粉蛋白的氨基酸序列等。而且，其可以含有衍生自具有其他病原性或變應(yīng)原性的蛋白的氨基酸序列。
例如，當(dāng)其含有衍生自HCV包膜蛋白的氨基酸序列時，可獲得治療，預(yù)防由HCV引起的疾病的HLA-結(jié)合肽。
<實施方案2>
根據(jù)該實施方案，提供了與HLA-A型分子結(jié)合的HLA-結(jié)合肽，含有在上述HLA-結(jié)合肽中所含的氨基酸序列基礎(chǔ)上通過缺失，替換，或添加一個或兩個氨基酸殘基而形成的氨基酸序列，并且由不少于8個且不多于11個氨基酸殘基組成。
如下文所述，即使組成包括在與HLA-A型分子結(jié)合的特異氨基酸序列中通過缺失，替換，或添加一個或數(shù)個氨基酸殘基而形成的氨基酸序列，仍顯示與上述實施方案1有關(guān)的HLA-結(jié)合肽相似的效果。
盡管上述HCV D90208株，D89815株，和pBRT703′X株(D89815的突變亞克隆)的多肽的氨基酸序列彼此部分相異，但由于D90208株某基因組蛋白中現(xiàn)有的若干9-聚體肽的-log Kd值的預(yù)測數(shù)據(jù)和實驗數(shù)據(jù)之間的相關(guān)性高，即，基于預(yù)測數(shù)據(jù)確定為結(jié)合的序列在實驗數(shù)據(jù)中顯示良好的-log Kd值，則可預(yù)測D89815株和pBRT703′X株(D89815的突變亞克隆)會顯示-log Kd值在預(yù)測數(shù)據(jù)中具有較好的名次排列。因此，可以預(yù)測，即使D89815株和pBRT703′X株(D89815的突變亞克隆)某些基因組蛋白中的氨基酸序列，是在顯示結(jié)合性質(zhì)的氨基酸序列的基礎(chǔ)上通過替換一個或兩個氨基酸殘基而形成的氨基酸序列，仍將同樣顯示優(yōu)異的HLA-結(jié)合性質(zhì)。
即，可以預(yù)測，即使氨基酸序列是在與HLA-A型分子結(jié)合方面具有優(yōu)異性質(zhì)的SEQ ID NOS1-83所示氨基酸序列的基礎(chǔ)上通過缺失，替換或添加一個或兩個氨基酸殘基而形成的，將以相似方式顯示優(yōu)異的HLA-結(jié)合性質(zhì)。
從另一觀點來看，可以預(yù)測，即使氨基酸序列是在通過上述方法預(yù)測在與HLA-A型分子結(jié)合方面具有優(yōu)異性質(zhì)的氨基酸序列的基礎(chǔ)上通過缺失，替換或添加一個或數(shù)個氨基酸殘基而形成的，仍會以相似方式顯示HLA-結(jié)合性質(zhì)。被替換的氨基酸殘基優(yōu)選為相互之間具有相似性質(zhì)的氨基酸殘基，例如，兩個皆為疏水性氨基酸殘基。
而且，利用本領(lǐng)域技術(shù)人員公知的方法，可以生產(chǎn)實施方案1和實施方案2中所述的HLA-結(jié)合肽。例如，所述結(jié)合肽可通過固相或液相方法人工合成?；蛘?，這些HLA-結(jié)合肽通過編碼這些HLA-結(jié)合肽的DNA區(qū)段或重組載體的表達(dá)而生產(chǎn)。所得的這些HLA-結(jié)合肽通過本領(lǐng)域技術(shù)人員公知的方法而鑒別。例如，采用Edman降解，質(zhì)譜等鑒別是可能的。
<實施方案3>
根據(jù)本實施方案，提供了含有為上述HLA-結(jié)合肽編碼的DNA序列的DNA區(qū)段。由于與本實施方案有關(guān)的DNA區(qū)段含有特異DNA序列，因此，其可表達(dá)上述HLA-結(jié)合肽。
當(dāng)利用與本實施方案有關(guān)的DNA區(qū)段表達(dá)上述HLA-結(jié)合肽時，通過將DNA區(qū)段導(dǎo)入細(xì)胞，可實現(xiàn)表達(dá)，或通過商用人工蛋白表達(dá)試劑盒，可實現(xiàn)表達(dá)。
此外，通過將上述DNA區(qū)段導(dǎo)入，例如人細(xì)胞，可實現(xiàn)連續(xù)表達(dá)。由此，通過將編碼HLA-結(jié)合肽的DNA區(qū)段導(dǎo)入細(xì)胞，而非將HLA-結(jié)合肽自身導(dǎo)入細(xì)胞，可使HLA-結(jié)合肽組成型存在于細(xì)胞中。當(dāng)HLA-結(jié)合肽用作疫苗時，這種組成型表達(dá)的能力對于提高疫苗的功效是有利的。
而且，與本實施方案有關(guān)的DNA區(qū)段可通過本領(lǐng)域技術(shù)人員公知的方法生產(chǎn)。例如，其可通過商用DNA合成儀等人工合成?；蛘?，其可通過限制性酶等從HCV基因組中片斷化。或者，其可利用引物對通過PCR方法從HCV基因組中擴增。所得的DNA區(qū)段利用本領(lǐng)域技術(shù)人員公知的方法而鑒別。例如，其可通過商用DNA測序儀而鑒別。
<實施方案4>
根據(jù)本實施方案，提供了含有為上述HLA-結(jié)合肽編碼的DNA序列的重組載體。由于與本實施方案有關(guān)的重組載體含有特異DNA序列，上述HLA-結(jié)合肽可得以表達(dá)。
當(dāng)上述HLA-結(jié)合肽通過與本實施方案有關(guān)的重組載體表達(dá)時，通過將該重組載體導(dǎo)入細(xì)胞可實現(xiàn)表達(dá)，或通過商用人工蛋白表達(dá)試劑盒可實現(xiàn)表達(dá)。
此外，通過將上述重組載體導(dǎo)入，例如人細(xì)胞，可實現(xiàn)連續(xù)表達(dá)。由此，通過將編碼HLA-結(jié)合肽的重組載體導(dǎo)入細(xì)胞，而非將HLA-結(jié)合肽自身導(dǎo)入細(xì)胞，可使HLA-結(jié)合肽連續(xù)存在于細(xì)胞中。當(dāng)HLA-結(jié)合肽用作疫苗時，這種連續(xù)表達(dá)的能力對提高疫苗的功效是有利的。
而且，在上述重組載體中，通過在參與轉(zhuǎn)錄和表達(dá)的調(diào)節(jié)區(qū)中使用某序列，例如位于編碼上述HLA-結(jié)合肽的DNA序列上游的啟動子區(qū)，HLA-結(jié)合肽的表達(dá)量可以高精度控制。而且，在參與復(fù)制的調(diào)節(jié)區(qū)中通過使用某序列，例如重組載體的起點區(qū)，重組載體在細(xì)胞中的拷貝數(shù)可得以高精度控制。
此外，上述重組載體可任選含有除編碼上述HLA-結(jié)合肽的NDA序列以外的序列。例如，其可含有標(biāo)記基因序列，諸如藥物抗性基因。
而且，與本實施方案有關(guān)的重組載體可利用本領(lǐng)域技術(shù)人員公知的方法生產(chǎn)。例如，其通過在某限制性酶切位點處切割商用載體，諸如pBR322或pUC19的多克隆位點，然后插入上述DNA區(qū)段至該位點，并連接而獲得。此外，所得重組載體利用本領(lǐng)域技術(shù)人員公知的方法可鑒別。例如，通過瓊脂糖凝膠電泳，可確認(rèn)由預(yù)定限制性酶切割的DNA區(qū)段的長度是否與商用載體，諸如pBR322或pUC19的限制性圖譜一致。此外，通過DNA測序儀等可鑒別上述DNA序列是否包含在從多克隆位點切割得到的DNA序列中。
盡管上文描述了本發(fā)明的實施方案，但它們僅列舉作為本發(fā)明的例子，除上述之外，可采用多種不同的變例。
例如，在上述實施方案中，HLA-結(jié)合肽含有衍生自HCV某基因組蛋白(SEQ ID NOS184，185，186)的氨基酸序列，但可采用含有衍生自另一HCV的氨基酸序列的HLA-結(jié)合肽。在這種情形下，所述結(jié)合肽可用于治療與其衍生的蛋白有關(guān)的多種免疫疾病。
此外，可采用除HCV以外的病原菌，諸如HIV病毒的HLA-結(jié)合肽，也可采用含有衍生自蛋白，諸如雪松花粉過敏原等，或癌細(xì)胞的氨基酸序列的HLA-結(jié)合肽。
這樣，如果含有通過上述方法預(yù)測具有優(yōu)異HLA-結(jié)合性質(zhì)的氨基序列，則可以預(yù)計，所述結(jié)合肽將顯示優(yōu)異的HLA-結(jié)合性質(zhì)，這與經(jīng)實驗確認(rèn)時的相似。由此，這些HLA-結(jié)合肽可合適地主要用于治療或預(yù)防傳染病(流感，SARS，HIV，HCV等)，并且也用于癌癥免疫治療，過敏性疾病(花粉過敏(花粉熱)，風(fēng)濕病，遺傳性過敏癥，哮喘等)，自身免疫疾病等。
(實施例)本發(fā)明下文將參照實施例進(jìn)一步解釋，但本發(fā)明并不限于此。
具體而言，基于積極學(xué)習(xí)實驗設(shè)計，實現(xiàn)本發(fā)明實施例中的預(yù)測，實驗和評價步驟，并且一般而言，重復(fù)下述步驟。在此采用的積極學(xué)習(xí)實驗設(shè)計的示意圖示于

圖1中。
(1)低級順序?qū)W習(xí)算法的試驗(將在下文描述)進(jìn)行一次。即，從累積數(shù)據(jù)中通過隨機采樣生成多個假設(shè)，關(guān)于隨機表達(dá)的候選查詢點(肽)，顯示預(yù)測值的最大分布的點挑選作為查詢點進(jìn)行實驗。
(2)被挑選的查詢點處的肽通過合成和純化方法(將在下文描述)而制備，以及實際結(jié)合能力通過實驗測量(將在下文描述)，并添加至累積數(shù)據(jù)。
在本發(fā)明實施例中，Hidden Markov模型的監(jiān)督學(xué)習(xí)算法用作低級順序?qū)W習(xí)算法，以223種肽的起始數(shù)據(jù)開始，根據(jù)實驗預(yù)測和挑選20-30種肽，上述步驟重復(fù)四次，獲得總共341個數(shù)據(jù)點。
更具體而言，在本發(fā)明實施例的積極學(xué)習(xí)方法中，根據(jù)實驗設(shè)計和合成了20-30種肽，在這些肽含有的氨基酸序列中，排列了9/20種氨基酸。測量了與HLA分子的結(jié)合強度(結(jié)合能力)。獲得的結(jié)合能力(Kd值，以摩爾濃度表示)作為實驗結(jié)果。當(dāng)結(jié)合能力高時，該肽被挑選作為HLA-結(jié)合肽的候選物，用作疫苗材料。
所得結(jié)果輸入裝配學(xué)習(xí)機的學(xué)習(xí)系統(tǒng)，采用Hidden Markov模型作為數(shù)學(xué)算法，并創(chuàng)建了規(guī)則。學(xué)習(xí)機采樣不同結(jié)果以制備規(guī)則。通過學(xué)習(xí)機表述的規(guī)則具有不同的組成。視需要，所得規(guī)則和實驗數(shù)據(jù)存儲作為累積數(shù)據(jù)。
從超過209＝5000億個肽序列中，通過規(guī)則挑選用于隨后實驗的候選物，并且重復(fù)上述步驟。在此階段，不同的規(guī)則適用于實驗候選物，以及實驗結(jié)果的預(yù)測產(chǎn)生分歧的候選物進(jìn)行實驗。這樣，由于實驗結(jié)果的預(yù)測產(chǎn)生分歧的候選物進(jìn)行隨后實驗，提高了預(yù)測的最終精度。
以此，多個學(xué)習(xí)機進(jìn)行選擇性采樣，其中給出不同預(yù)測的樣品被挑選作為實驗候選物，可有效增加信息，以及獲得高精度的假設(shè)(規(guī)則)。重復(fù)上述步驟四次，給出優(yōu)異結(jié)果，如下文實施例所述。重復(fù)7次或以上給出甚至更好的結(jié)果。
根據(jù)這種積極學(xué)習(xí)方法，可減少由9個氨基酸殘基組成的肽的結(jié)合實驗的重復(fù)次數(shù)，其本應(yīng)對HLA-結(jié)合肽的所有候選物進(jìn)行5000億或以上的組合。在積極學(xué)習(xí)方法中，通過實驗形成規(guī)則，對數(shù)十個通過應(yīng)用規(guī)則預(yù)測的候選序列重復(fù)實驗。由此，實驗次數(shù)可削減，初始篩選的時間和費用大大減少。
此外，采用規(guī)則預(yù)測肽與HLA結(jié)合的命中率可達(dá)70-80％，所述規(guī)則通過積極學(xué)習(xí)方法獲得，而通過其他公知技術(shù)，諸如錨定方法得到的命中率低至約30％。
<肽的合成和純化>
利用Fmoc氨基酸，通過Merrifield固相方法，手工合成肽。脫保護(hù)之后，利用C18柱進(jìn)行反相HPLC純化，給出95％或以上的純度。采用MALDI-TOF質(zhì)譜儀(Voyager DE RP，PerSeptive)，鑒別肽和確認(rèn)其純度。通過Micro BCA分析(Pierce Corp.)，利用BSA作為標(biāo)準(zhǔn)蛋白，進(jìn)行肽的定量分析。
<肽與HLA-A24型分子結(jié)合的實驗>
肽與HLA-A24型分子(HLA-A*2402基因的產(chǎn)物)結(jié)合的能力，利用表達(dá)HLA-A24型分子的C1R-A24細(xì)胞(由熊本大學(xué)的MasafumiTakiguchi教授制備的細(xì)胞，在征得其同意下，通過Ehime University的Masaki Yasukawa助理教授提供)，加以測量。
C1R-A24細(xì)胞首先暴露于酸性條件，pH3.3，30秒，由此解離和去除輕鏈β2m(其與HLA I型分子共同相連)，以及原先與HLA-A*2402分子結(jié)合的內(nèi)源肽。中和后，純化的β2m加至C1R-A24細(xì)胞，所得產(chǎn)物添加至系列稀釋的肽中，冰上溫育4小時。利用熒光標(biāo)記的單克隆抗體17A12染色，所述抗體識別HLA-A*2402分子，肽，和β2m三個成員的結(jié)合(MHC-pep)，這三個成員在溫育過程中已重新結(jié)合。隨后，MHC-pep計數(shù)/C1R-A24細(xì)胞(與上述熒光抗體的熒光強度成比例)利用FACScan熒光活化的細(xì)胞分揀儀(Becton DickinsonBiosciences)定量測量。HLA-A24型分子和肽之間的結(jié)合解離常數(shù)Kd值，通過公開的方法(Udaka等，Immunogenetics，51，816-828，2000)，根據(jù)每個細(xì)胞的平均熒光強度而計算。
<肽與HLA-A2型分子結(jié)合的實驗>
肽與HLA-A2型分子(HLA-A*0201基因的產(chǎn)物)結(jié)合的能力，利用表達(dá)HLA-A*0201的株JY細(xì)胞，加以測量。
JY細(xì)胞首先暴露于酸性條件，pH3.8，30秒，由此解離和去除與HLA-A*0201分子非共價連接的輕鏈β2m和內(nèi)源肽。中和后，進(jìn)行重新結(jié)合實驗。
上述JY細(xì)胞和純化的β2m加至分級系列稀釋的結(jié)合能力被測量的肽，冰上溫育4小時。利用結(jié)合型特異的熒光標(biāo)記的單克隆抗體BB7.2，對已重新結(jié)合高達(dá)該點的HLA-A*0201分子染色。
隨后，每個細(xì)胞的熒光量，利用流式細(xì)胞儀測量，而解離常數(shù)Kd值，通過公開的方法(Udaka等，Immunogenetics，51，816-828，2000)，以摩爾濃度計算。
<評價結(jié)果>
獲得的預(yù)測結(jié)果和實驗結(jié)果示于上文表1-4中。
表1中的序列SEQ ID NOS1-44由登記在GenBank中的HCVD90208株某基因組蛋白的全長序列中所含的9個氨基酸殘基組成。此外，序列SEQ ID NOS1-44具有較好的與HLA-A24型分子結(jié)合，正如通過假設(shè)預(yù)測，所述假設(shè)由實施方案1中所述的實驗設(shè)計方法獲得。SEQ ID NOS1-44以結(jié)合降低的順序排列。即，SEQ ID NO1為預(yù)測具有最佳結(jié)合的序列。HCV D90208某基因組蛋白的全長氨基酸序列示于下列SEQ ID NO184(MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGN
EGMGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTIPASAYEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGCVPCVRESNFSRCWVALTPTLAARNSSIPTTTIRRHVDLLVGAAALCSAMYVGDLCGSVFLVSQLFTFSPRRYETVQDCNCSIYPGHVSGHRMAWDMMMNWSPTTALVVSQLLRIPQAVVDMVAGAHWGVLAGLAYYSMVGNWAKVLIVMLLFAGVDGHTHVTGGRVASSTQSLVSWLSQGPSQKIQLVNTNGSWHINRTALNCNDSLQTGFIAALFYAHRFNASGCPERMASCRPIDEFAQGWGPITHDMPESSDQRPYCWHYAPRPCGIVPASQVCGPVYCFTPSPVVVGTTDRFGAPTYSWGENETDVLLLSNTRPPQGNWFGCTWMNSTGFTKTCGGPPCNIGGVGNNTLVCPTDCFRKHPEATYTKCGSGPWLTPRCMVDYPYRLWHYPCTVNFTVFKVRMYVGGVEHRLNAACNWTRGERCDLEDRDRSELSPLLLSTTEWQILPCSFTTLPALSTGLIHLHRNIVDVQYLYGIGSAVVSFAIKWEYILLLFLLLADARVCACLWMMLLIAQAEATLENLVVLNAASVAGAHGLLSFLVFFCAAWYIKGRLVPGAAYALYGVWPLLLLLLALPPRAYAMDREMAASCGGAVFVGLVLLTLSPYYKVFLARLIWWLQYFITRAEAHLQVWVPPLNVRGGRDAIILLTCAVHPELIFDITKLLLAILGPLMVLQAGITRVPYFVRAQGLIRACMLVRKVAGGHYVQMAFMKLAALTGTYVYDHLTPLRDWAHAGLRDLAVAVEPVVFSDMETKLITWGADTAACGDIISGLPVSARRGKEILLGPADSFGEQGWRLLAPITAYSQQTRGLLGCIITSLTGRDKNQVDGEVQVLSTATQSFLATCVNGVCWTVYHGAGSKTLAGPKGPITQMYTNVDQDLVGWPAPPGARSMTPCTCGSSDLYLVTRHADVVPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPSGHVVGIFRAAVCTRGVAKAVDFIPVESMETTMRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIEPNIRTGVRTITTGGPITYSTYCKFLADGGCSGGAYDIIICDECHSTDSTTILGIGTVLDQAETAGARLVVLATATPPGSITVPHPNIEEVALSNTGEIPFYGKAIPIEAIKGGRHLIFCHSKKKCDELAAKLTGLGLNAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIET
TTLPQDAVSRAQRRGRTGRGRSGIYRFVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETSVRLRAYLNTPGLPVCQDHLEFWESVFTGLTHIDAHFLSQTKQAGDNLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMACMSADLEVVTSTWVLVGGVLAALAAYCLTTGSVVIVGRIILSGRPAVIPDREVLYQEFDEMEECASHLPYIEQGMQLAEQFKQKALGLLQTATKQAEAAAPVVESKWRALEVFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTASITSPLTTQNTLLFNILGGWVAAQLAPPSAASAFVGAGIAGAAVGSIGLGKVLVDILAGYGAGVAGALVAFKVMSGEMPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSSLTITQLLKRLHQWINEDCSTPCSGSWLKDVWDWICTVLSDFKTWLQSKLLPRLPGLPFLSCQRGYKGVWRGDGIMQTTCPCGAQITGHVKNGSMRIVGPKTCSNTWHGTFPINAYTTGPCTPSPAPNYSRALWRVAAEEYVEVTRVGDFHYVTGMTTDNVKCPCQVPAPEFFTEVDGVRLHRYAPVCKPLLREEVVFQVGLNQYLVGSQLPCEPEPDVAVLTSMLTDPSHITAETAKRRLARGSPPSLASSSASQLSAPSLKATCTTHHDSPDADLIEANLLWRQEMGGNITRVESENKVVILDSFDPIRAVEDEREISVPAEILRKPRKFPPALPIWARPDYNPPLLESWKDPDYVPPVVHGCPLPSTKAPPIPPPRRKRTVVLTESTVSSALAELATKTFGSSGSSAVDSGTATGPPDQASDDGDKGSDVESYSSMPPLEGEPGDPDLSDGSWSTVSGEAGEDVVCCSMSYTWTGALITPCAAEESKLPINPLSNSLLRHHSMVYSTTSRSASLRQKKVTFDRLQVLDDHYRDVLKEMKAKASTVKARLLSIEEACKLTPPHSAKSKFGYGAKDVRSLSSRAVNHIRSVWEDLLEDTETPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSTLPQAVMGPSYGFQYSPGQRVEFLVNTWKSKKCPMGFSYDTRCFDSTVTENDIRTEESIYQCCDLAPEARQAIRSLTERLYVGGPLTNSKGQNCGYRRCRASGVLTTSCGNTLTCYLKATAACRAAKLQDCTMLVNGDDLVVICESAGTQEDAAALRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETVRHTPVNSWLGNIIMYAPTLWARMILMTHFFSILLAQEQLEKALDCQIYG
ACYSIEPLDLPQIIERLHGLSAFSLHSYSPGEINRVASCLRKLGVPPLRVWRHRARSVRAKLLSQGGRAATCGKYLFNWAVKTKLKLTPIPAASQLDLSGWFVAGYNGGDIYHSLSRARPRWFMLCLLLLSVGVGIYLLPNR).
序列SEQ ID NOS45-83由登記在GenBank中的HCV D89815株某基因組蛋白的全長序列中所含的9個氨基酸殘基組成。此外，序列SEQ ID NOS45-83具有較好的與HLA-A24型分子結(jié)合，正如通過假設(shè)預(yù)測，所述假設(shè)由實施方案1中所述的實驗設(shè)計方法獲得。SEQ IDNOS45-83以結(jié)合降低的順序排列。即，SEQ ID NO45為預(yù)測具有最佳結(jié)合的序列。HCV D89815株某基因組蛋白的全長氨基酸序列示于下列SEQ ID NO185(MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGLGWAGWLLSPRGSRPSWGPNDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTIPASAYEVRNVSGIYHVTNDCSNSSIVYEAADVIMHAPGCVPCVRENNSSRCWVALTPTLAARNASVPTTTLRRHVDLLVGTAAFCSAMYVGDLCGSVFLISQLFTFSPRRHETVQDCNCSIYPGHVSGHRMAWDMMMNWSPTAALVVSQLLRIPQAVMDMVAGAHWGVLAGLAYYSMVGNWAKVLIVMLLFAGVDGHTRVTGGVQGHVTSTLTSLFRPGASQKIQLVNTNGSWHINRTALNCNDSLKTGFLAALFYTHKFNASGCPERMASCRSIDKFDQGWGPITYAQPDNSDQRPYCWHYAPRQCGIVPASQVCGPVYCFTPSPVVVGTTDRFGAPTYNWGDNETDVLLLNNTRPPHGNWFGCTWMNSTGFTKTCGGPPCNIRGVGNNTLTCPTDCFRKHPDATYTKCGSGPWLTPRCLVDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRLDAACNWTRGERCDLEDRDRAELSPLLLSTTEWQILPCSYTTLPdALSTGLIHLHQNIVDIQYLYGIGSAVVSIAIKWEYVVLLFLLLADARVCACLWMMLLIAQAEAALENLVVLNAASVVGAHGMLPFFMFFCAAWYMKGRLVPGAAYAFYGVWPLLLLLLALPPRAYAMDREMVASCG
GGVFVGLALLTLSPYCKVFLARLIWWLQYFITKAEAHLQVSLPPLNVRGGRDAIILLMCAVHPELIFDITKLLLSILGPLMVLQASLIRVPYFVRAQGLIRACMLVRKAAGGHYVQMAFVKLAALTGTYVYDHLTPLQDWAHVGLRDLAVAVEPVVFSAMETKVITWGADTAACGDIISGLPVSARRGKEILLGPADSFEGQGWRLLAPITAYSQQTRGLLGCIITSLTGRDKNQVEGEVQVVSTAKQSFLATCVNGACWTVFHGAGSKTLAAAKGPITQMYTNVDQDLVGWPAPPGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPSGHVVGIFRAAVCTRGVAKAVDFIPVESMETTMRSPVFTDNSTPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYMVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGAPITYSTYGKFLADGGCSGGAYDIIICDECHSTDSTSILGIGTVLDQAETVGARFVVLATATPPGSITFPHPNIEEVPLANTGEIPFYAKTIPIEVIRGGRHLIFCHSKKKCDELPAKLSALGLNAVAYYRGLDVSVIPASGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTVPQDAVSRTQRRGRTGRGRRGIYRFVTPGERPSAMFDSSVLCECYDAGCAWYELTPAETSVRLRAYLNTPGLPVCQDHLEFWESVFTGLTHIDAHFLSQTKQAGDNFPYLVAYQATVCARAKAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMACMSADLEVVTSTWVLVGGVLAALAAYCLTTGSVVIVGRIILSGRPAVIPDREVLYQEFDEMEECASHLPYIEQGMQLAEQFKQKALGLLQTATKQAEAAAPVVESKWRALETFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTASITSPLATQYTLLFNILGGWVAAQLAPPSAASAFVGAGIAGAAVGSIGLGKVLVDILAGYGAGVAGALVAFKVMSGDMPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSNLTITQLLKRLHQWINEDCSTPCSGSWLRDVWDWICTVLADFKTWLQSKLLPRLPGVPFFSCQRGYKGVWRGDGIMYTTCPCGAQITGHVKNGSMRIVGPRTCSNTWHGTFPINAYTTGPCTPSPAPNYSRALWRVAAEEYVEVTRVGDFHYVTGMTTDNVKCPCQVPAPEFFTELDGVRLHRYAPACKPLLRDEVTFQVGLNQYTVGSQLPCEPEPDVTVVTSMLTDPSHITAEAARRRLARGSPPSLAGSSASQLSALSLKATCT
THHGAPDTDLIEANLLWRQEMGGNITRVESENKIVILDSFEPLRAEEDEREVSAAAEILRKTRKFPAAMPVWARPDYNPPLLESWKNPDYVPPVVHGCPLPPTKAPPIPPPRRKRTVVLTESTVSSALAELATKTFGGSGSSAVDSGTATGPPDQASAEGDAGSDAESYSSMPPLEGEPGDPDLSDGSWSTVSEEASEDVVCCSMSYTWTGALITPCAAEESKLPINALSNPLLRHHNMVYSTTSRSASLRQKKVTFDRMQVLDDHYRDVLKEMKAKASTVKAKLLSVEEACKLTPPHSAKSKFGYGAKDVRSLSSRAVNHIRSVWKDLLEDTDTPIQTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSTLPQAVMGSSYGFQYSPKQRVEFLVNTWKAKKCPMGFSYDTRCFDSTVTENDIRVEESIYQCCDLAPEARQAIRSLTERLYIGGPMTNSKGQNCGYRRCRASGVLTTSCGNTLTCYLKAAAACRAAKLQDCTMLVCGDDLVVICDSAGTQEDAASLRVFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMYAPTLWARMILMTHFFSILLAQEQLEKALDCQIYGATYSIEPLDLPQIIQRLHGLSAFSLHSYSPGEINRVASCLRKLGVPPLRVWRHRARSVRAKLLSQGGRAATCGKYLFNWAVKTKLKLTPIPEASQLDLSGWFVAGYSGGDIYHSLSRARPRWFMWCLLLLSVGVGIYLLPNR).
序列SEQ ID NOS84-123由HCV pBRT703′X株(D89815的突變亞克隆)某基因組蛋白中所含的9個氨基酸殘基組成，所述毒株獲自大阪大學(xué)微生物疾病研究所的Yoshiharu Matsuura教授。此外，序列SEQ IDNOS84-123具有較好的與HLA-A24型分子結(jié)合，正如通過假設(shè)預(yù)測，所述假設(shè)由實施方案1中所述的實驗設(shè)計方法獲得。SEQ IDNOS84-123以結(jié)合降低的順序排列。即，SEQ ID NO84為預(yù)測具有最佳結(jié)合的序列。HCV pBRT703′X株(D89815的突變亞克隆)某基因組蛋白的全長氨基酸序列示于下列SEQ ID NO186(MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARHPEGRAWAQPGYPWPLYGNEGMGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADL
MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTIPASAYEVRNVSGIYHVTNDCSNSSIVYEAADVIMHAPGCVPCVRENNSSRCWVALTPTLAARNASVPTTTLRRHVDLLVGTAAFCSAMYVGDLCGSVFLISQLFTFSPRRHETVQDCNCSIYPGHVSGHRMAWDMMMNWSPTAALVVSQLLRIPQAVMDMVAGAHWGVLAGLAYYSMVGNWAKVLIVMLLFAGVDGHTRVTGGVQGHVTSTLTSLFRPGASQKIQLVNTNGSWHINRTALNCNDSLKTGFLAALFYTHKFNASGCPERMASCRSIDKFDQGWGPITYAQPDNSDQRPYCWHYAPRQCGIVPASQVCGPVYCFTPSPVVVGTTDRFGAPTYNWGDNETDVLLLNNTRPPHGNWFGCTWMNSTGFTKTCGGPPCNIRGVGNNTLTCPTDCFRKHPDATYTKCGSGPWLTPRCLVDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRLDAACNWTRGERCDLEDRDRAELSPLLLSTTEWQILPCSYTTLPALSTGLIHLHQNIVDIQYLYGIGSAVVSIAIKWEYVVLLFLLLADARVCACLWMMLLIAQAEAALENLVVLNAASVAGAHGILPFFMFFCAAWYMKGRLVPGAAYAFYGVWPLLLLLLALPPRAYAMDREMAASCGGGVFVGLALLTLSPYCKVFLARLIWWLQYFITKAEAHLQVWVPPLNVRAGRDAIILLMCAVHPELIFDITKLLLSILGPLMVLQASLIRVPYFVRAQGLIRACTLVRKAAGGHYVQMAFVKLAALTGTYVYDHLTPLQDWAHVGLRDLAVAVEPVVFSAMETKVITWGADTAACGDIISGLPVSARRGKEILLGPADSFEGQGWRLLAPITAYSQQTRGLLGCIITSLTGRDKNQVEGEVQVVSTATQSFLATCVNGACWTVFHGAGSKTLAGPKGPITQMYTNVDQDLVGWPAPPGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDTRGSLLSPRPISYLKGSSGGPLLCPSGHVVGIFRAAVCTRGVAKAVDFIPVESMETTMRSPVFTDNSTPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYMVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGAPITYSTYGKFLADGGCSGGAYDIIICDECHSTDSTSILGIGTVLDQAETAGARLVVLATATPPGSVTFPHPNIEEVALGNTGEIPFYGKAIPIEVIKGGRHLIFCHSKKKCDELAAKLSPLGLNAVAYYRGLDVSVIPASGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTVPQDAVSRTQRRGRTGRGRRGIYRFVTPGERPSGMFDSSVLCECYD
AGCAWYELTPAETSVRLRAYLNTPGLPVCQDHLEFWESVFTGLTHIDAHFLSQTKQAGDNFPYLVAYQATVCARAKAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKFIMACMSADLEVVTSTWVLVGGVLAALAAYCLTTGSVVIVGRIILSGRPAVIPDREVLYQEFDEMEECASHLPYIEQGMQLAEQFKQKALGLLQTATKQAEAAAPVVESKWRALETFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTASITSPLATQYTLLFNILGGWVAAQLAPPSAASAFVGAGIAGAAVGSIGLGKVLVDILAGYGAGVAGALVAFKVMSGDMPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSNLTITQLLKRLHQWINEDCSTPCSGSWLRDVWDWICTVLADFKTWLQSKLLPRLPGVPFFSCQRGYKGVWRGDGIMYTTCPCGAQITGHVKNGSMRIVGPRTCSNTWHGTFPINAYTTGPCTPSPAPNYSRALWRVAAEEYVEVTRVGDFHYVTGMTTDNVKCPCQVPAPEFFTELDGVRLHRYAPACKPLLRDEVTFQVGLNQYTVGSQLPCEPEPDVTVVTSMLTDPSHITAEAARRRLARGSPPSLAGSSASQLSAPSLKATCTTHHGAPDTDLIEANLLWRQEMGGNITRVESENKIVILDSFEPLRAEEDEREVSAAAEILRKTRKFPAAMPVWARPDYNPPLLESWKNPDYVPPVVHGCPLPPTKAPPIPPPRRKRTVVLTESTVSSALAELATKTFGGSGSSAVDSGTATGPPDQASAEGDAGSDAESYSSMPPLEGEPGDPDLSDGSWSTVSEEASEDVVCCSMSYTWTGALITPCAAEESKLPINALSNPLLRHHNMVYSTTSRSASLRQKKVTFDRMQVLDDHYRDVLKEMKAKASTVKAKLLSVEEACKLTPPHSAKSKFGYGAKDVRSLSSRAVNHIRSVWKDLLEDTDTPIQTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSTLPQAVMGSSYGFQYSPKQRVEFLVNTWKAKKCPMGFSYDTRCFDSTVTENDIRVEESIYQCCDLAPEARQAIRSLTERLYIGGPMTNSKGQNCGYRRCRASGVLTTSCGNTLTCYLKAAAACRAAKLQDCTMLVCGDDLVVICDSAGTQEDAASLRVFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMYAPTLWARMILMTHFFSILLAQEQLEKALDCQIYGATYSIEPLDLPQIIQRLHGLSAFSLHSYSPGEINRVASCLRKLGVPP
LRVWRHRARSVRAKLLSQGGRAATCGKYLFNWAVKTKLKLTPIPEASQLDLSGWFVAGYSGGDIYHSLSRARPRWFMWCLLLLSVGVGIYLLPNR).
序列SEQ ID NOS124-183由上述HCV pBRT703′X株(D89815的突變亞克隆)某基因組蛋白中所含的9個氨基酸殘基組成。此外，序列SEQ ID NOS124-183具有較好的與HLA-A2型分子結(jié)合，正如通過假設(shè)預(yù)測，所述假設(shè)由實施方案1中所述的實驗設(shè)計方法獲得。SEQ IDNOS124-183以結(jié)合降低的順序排列。即，SEQ ID NO124為預(yù)測具有最佳結(jié)合的序列。
表1-4顯示的氨基酸序列在對HCV D90208株，D89815株，和pBRT703′X株(D89815的突變亞克隆)，利用上述預(yù)測程序，預(yù)測分，和相應(yīng)結(jié)合實驗數(shù)據(jù)而獲得的預(yù)測結(jié)果中具有較好的得分。通過上述合成方法人工合成9個氨基酸肽，可獲得全部結(jié)合實驗數(shù)據(jù)。
所述的HCV D90208株和D89815株的某些基因組蛋白登記在GenBank，但由其中9個氨基酸殘基組成的序列，即HLA-結(jié)合肽，現(xiàn)在尚未登記。
此外，pBRT703′X株(D89815的突變亞克隆)為突變株，其與日本人丙型肝炎病人中常見的HCV亞株相似。在本發(fā)明實施例中，已發(fā)現(xiàn)在該突變株某基因組蛋白中所含的HLA-結(jié)合肽，所述突變株與常見于日本人中的亞株相似。該HLA-結(jié)合肽可合適地用于開發(fā)日本人的丙型肝炎治療藥。
在此，上述HCV D90208株，D89815株，和pBRT703′X株(D89815的突變亞克隆)某些基因組蛋白的氨基酸序列彼此部分不同，并且可以預(yù)測，即使在氨基酸序列中通過替換一個或數(shù)個氨基酸殘基而形成的氨基酸序列將同樣顯示上述優(yōu)異的HLA-結(jié)合性質(zhì)。
例如，D90208株的肽SEQ ID NO1從左計數(shù)第6個氨基酸為F，但其對D89815株的肽SEQ ID NO46和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO85為Y。
此外，D90208株的肽SEQ ID NO17從左計數(shù)第2個氨基酸為L，但其對D89815株的肽SEQ ID NO49和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO98為F。
而且，D90208株的肽SEQ ID NO3從左計數(shù)第5個氨基酸為V，但其對D89815株的肽SEQ ID NO71和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO110為A。
此外，D90208株的肽SEQ ID NO2從左計數(shù)第7個氨基酸為D，但其對D89815株的肽SEQ ID NO45和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO84為E。
而且，D90208株的肽SEQ ID NO40從左計數(shù)第7個氨基酸為C，但其對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO107為G。
此外，D90208株的肽SEQ ID NO43從左計數(shù)第5個氨基酸為E，但其對D89815株的肽SEQ ID NO59和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO87為D，以及D90208株的肽SEQ ID NO43從左計數(shù)第8個氨基酸為V，但其對D89815株的肽SEQ ID NO59和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO87為T。
而且，D90208株的肽SEQ ID NO44從左計數(shù)第7個氨基酸為I，但其對D89815株的肽SEQ ID NO62和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO93為V，以及D90208株的肽SEQ ID NO44從左計數(shù)第9個氨基酸為V，但其對D89815株的肽SEQ ID NO62和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO93為F。
在通過相互替換一個或兩個氨基酸殘基而形成的肽序列中，例如，D90208株的肽SEQ ID NO17從左計數(shù)第2個氨基酸為L，但其對D89815株的肽SEQ ID NO49和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO98為F，以及D90208株的肽SEQ ID NO17的結(jié)合實驗值為6.98038，而其對D89815株的肽SEQ ID NO49和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO98為7.7344，由此證實它們?nèi)匡@示良好的結(jié)合性質(zhì)。
此外，在通過相互替換一個或兩個氨基酸殘基而形成的肽序列中，例如，D90208株的肽SEQ ID NO40從左計數(shù)第7個氨基酸為C，但其對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO107為G，以及D90208株的肽SEQ ID NO40的結(jié)合實驗值為6.97507，而其對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO107為6.37373，由此證實它們?nèi)匡@示良好的結(jié)合性質(zhì)。
而且，在通過相互替換一個或兩個氨基酸殘基而形成的肽序列中，例如，D90208株的肽SEQ ID NO3從左計數(shù)第5個氨基酸為V，但其對D89815株的肽SEQ ID NO71和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO110為A，以及D90208株的肽SEQ ID NO3的結(jié)合實驗值為6.14848，而其對D89815株的肽SEQ ID NO71和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO110為6.75756，由此證實它們?nèi)匡@示良好的結(jié)合性質(zhì)。
此外，在通過相互替換一個或兩個氨基酸殘基而形成的肽序列中，例如，D90208株的肽SEQ ID NO2從左計數(shù)第7個氨基酸為D，但其對D89815株的肽SEQ ID NO45和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO84為E，以及D90208株的肽SEQ ID NO2的結(jié)合實驗值為5.32417，而其對D89815株的肽SEQ ID NO45和對pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO84為5.00343，由此證實它們?nèi)匡@示良好的結(jié)合性質(zhì)。
而且，在通過相互替換一個或兩個氨基酸殘基而形成的肽序列中，例如，在pBRT703′X株(D89815的突變亞克隆)的肽SEQ ID NO90和肽SEQ ID NO112之間偏移一個的氨基酸序列，以及肽SEQ ID NO90的結(jié)合實驗值為6.51874，而肽SEQ ID NO112的結(jié)合實驗值為6.63423，由此證實它們?nèi)匡@示良好的結(jié)合性質(zhì)。
此外，在通過相互替換一個或兩個氨基酸殘基而形成的肽序列中，例如，在D90208株的肽SEQ ID NO13和D89815株的肽SEQ ID NO60與D90208株的肽SEQ ID NO18和D89815株的肽SEQ ID NO64之間偏移一個的氨基酸序列，以及肽SEQ ID NOS13和60的結(jié)合實驗值為7.89519，而肽SEQ ID NOS18和64的結(jié)合實驗值為5.9169，由此證實它們?nèi)匡@示良好的結(jié)合性質(zhì)。
由此可以預(yù)測，通過相互替換一個或兩個氨基酸殘基而形成的肽序列皆會顯示優(yōu)異的與HLA-A24型分子的結(jié)合?？傊?，由SEQ IDNOS1-183所示，在與HLA-A型分子結(jié)合方面具有優(yōu)異性質(zhì)的氨基酸序列中，通過缺失，替換，或添加一個或數(shù)個氨基酸殘基而形成的氨基酸序列，甚至同樣可以預(yù)測顯示優(yōu)異的HLA-結(jié)合性質(zhì)。
從另一點來看，正如通過假設(shè)預(yù)測，所述假設(shè)由實施方案1中所述的實驗設(shè)計方法獲得，在與HLA-A型分子結(jié)合方面具有優(yōu)異性質(zhì)的氨基酸序列中，通過缺失，替換，或添加一個或數(shù)個氨基酸殘基而形成的氨基酸序列，甚至同樣可以說顯示優(yōu)異的HLA-結(jié)合性質(zhì)。被替換的氨基酸殘基優(yōu)選為相互具有相似性質(zhì)的氨基酸殘基，諸如兩個皆為疏水氨基酸殘基。
本發(fā)明參照上述實施例進(jìn)行了解釋。這些實施例僅列舉成例子，本領(lǐng)域技術(shù)人員將理解，多種修改例是可能的，而這些修改例仍落入本發(fā)明的保護(hù)范圍。
例如，在上述實施例中，采用HCV D90208株，D89815株，和pBRT703′X株(D89815的突變亞克隆)，但也可采用另一HCV株。在此情形下，根據(jù)本發(fā)明所用的預(yù)測程序，可以高精度預(yù)測HLA結(jié)合。
序列表<110>日本電氣株式會社(NEC Corporation)國立大學(xué)法人高知大學(xué)(Kochi University)<120>HLA-結(jié)合肽，其前體，編碼這些肽序列的DNA片段和重組載體(HLA binding peptides)<130>SCT064984-47<160>186<170>PatentIn version 3.1<210>1<211>9<212>PRT<213>Hepatitis C virus<400>1Ile Leu Pro Cys Ser Phe Thr Thr Leu1 5<210>2<211>9<212>PRT<213>Hepatitis C virus<400>2Val Ile Leu Asp Ser Phe Asp Pro Ile1 5<210>3<211>9<212>PRT<213>Hepatitis C virus<400>3Arg Tyr Ala Pro Val Cys Lys Pro Leu1 5<210>4<211>9<212>PRT<213>Hepatitis C virus
<400>4Phe Trp Ala Lys His Met Trp Asn Phe1 5<210>5<211>9<212>PRT<213>Hepatitis C virus<400>5Ala Leu Tyr Asp Val Val Ser Thr Leu1 5<210>6<211>9<212>PRT<213>Hepatitis C virus<400>6Thr Val Leu Ser Asp Phe Lys Thr Trp1 5<210>7<211>9<212>PRT<213>Hepatitis C virus<400>7Pro Tyr Ile Glu Gln Gly Met Gln Leu1 5<210>8<211>9<212>PRT<213>Hepatitis C virus<400>8Trp His Tyr Pro Cys Thr Val Asn Phe1 5<210>9
<211>9<212>PRT<213>Hepatitis C virus<400>9Lys Phe Pro Pro Ala Leu Pro Ile Trp1 5<210>10<211>9<212>PRT<213>Hepatitis C virus<400>10Thr Tyr Ser Thr Tyr Cys Lys Phe Leu1 5<210>11<211>9<212>PRT<213>Hepatitis C virus<400>11Ala Tyr Ser Gln Gln Thr Arg Gly Leu1 5<210>12<211>9<212>PRT<213>Hepatitis C virus<400>12Ala Gln Pro Gly Tyr Pro Trp Pro Leu1 5<210>13<211>9<212>PRT<213>Hepatitis C virus<400>13Ile Leu Met Thr His Phe Phe Ser Ile
15<210>14<211>9<212>PRT<213>Hepatitis C virus<400>14Ser Tyr Thr Trp Thr Gly Ala Leu Ile1 5<210>15<211>9<212>PRT<213>Hepatitis C virus<400>15Ser Pro Pro Ala Val Pro Gln Thr Phe1 5<210>16<211>9<212>PRT<213>Hepatitis C virus<400>16Leu Leu Pro Arg Arg Gly Pro Arg Leu1 5<210>17<211>9<212>PRT<213>Hepatitis C virus<400>17Ala Leu Tyr Gly Val Trp Pro Leu Leu1 5<210>18<211>9<212>PRT<213>Hepatitis C virus
<400>18Leu Met Thr His Phe Phe Ser Ile Leu1 5<210>19<211>9<212>PRT<213>Hepatitis C virus<400>19Leu Leu Lys Arg Leu His Gln Trp Ile1 5<210>20<211>9<212>PRT<213>Hepatitis C virus<400>20Tyr Ile Leu Leu Leu Phe Leu Leu Leu1 5<210>21<211>9<212>PRT<213>Hepatitis C virus<400>21Ala Arg Pro Asp Tyr Asn Pro Pro Leu1 5<210>22<211>9<212>PRT<213>Hepatitis C virus<400>22Ala Tyr Tyr Ser Met Val Gly Asn Trp1 5
<210>23<211>9<212>PRT<213>Hepatitis C virus<400>23Phe Leu Ala Arg Leu Ile Trp Trp Leu1 5<210>24<211>9<212>PRT<213>Hepatitis C virus<400>24Ser Gln Leu Asp Leu Ser Gly Trp Phe1 5<210>25<211>9<212>PRT<213>Hepatitis C virus<400>25Ser Met Leu Thr Asp Pro Ser His Ile1 5<210>26<211>9<212>PRT<213>Hepatitis C virus<400>26Glu Tyr Ile Leu Leu Leu Phe Leu Leu1 5<210>27<211>9<212>PRT<213>Hepatitis C virus<400>27
Ile Leu Leu Gly Pro Ala Asp Ser Phe1 5<210>28<211>9<212>PRT<213>Hepatitis C virus<400>28Leu Asn Pro Ser Val Ala Ala Thr Leu1 5<210>29<211>9<212>PRT<213>Hepatitis C virus<400>29Gly Leu Leu Ser Phe Leu Val Phe Phe1 5<210>30<211>9<212>PRT<213>Hepatitis C virus<400>30Tyr Val Tyr Asp His Leu Thr Pro Leu1 5<210>31<211>9<212>PRT<213>Hepatitis C virus<400>31His Tyr Ala Pro Arg Pro Cys Gly Ile1 5<210>32<211>9<212>PRT
<213>Hepatitis C virus<400>32Gly Leu Ile His Leu His Arg Asn Ile1 5<210>33<211>9<212>PRT<213>Hepatitis C virus<400>33His Tyr Arg Asp Val Leu Lys Glu Met1 5<210>34<211>9<212>PRT<213>Hepatitis C virus<400>34Tyr Tyr Lys Val Phe Leu Ala Arg Leu1 5<210>35<211>9<212>PRT<213>Hepatitis C virus<400>35Cys Met Val Asp Tyr Pro Tyr Arg Leu1 5<210>36<211>9<212>PRT<213>Hepatitis C virus<400>36Ala Val Ile Pro Asp Arg Glu Val Leu1 5
<210>37<211>9<212>PRT<213>Hepatitis C virus<400>37Asn Phe Ser Arg Cys Trp Val Ala Leu1 5<210>38<211>9<212>PRT<213>Hepatitis C virus<400>38Val Phe Ser Asp Met Glu Thr Lys Leu1 5<210>39<211>9<212>PRT<213>Hepatitis C virus<400>39Val Trp Pro Leu Leu Leu Leu Leu Leu1 5<210>40<211>9<212>PRT<213>Hepatitis C virus<400>40Ile Thr Tyr Ser Thr Tyr Cys Lys Phe1 5<210>41<211>9<212>PRT<213>Hepatitis C virus<400>41
Ile Glu Pro Leu Asp Leu Pro Gln Ile1 5<210>42<211>9<212>PRT<213>Hepatitis C virus<400>42Leu Leu Ser Thr Thr Glu Trp Gln Ile1 5<210>43<211>9<212>PRT<213>Hepatitis C virus<400>43Pro Leu Leu Arg Glu Glu Val Val Phe1 5<210>44<211>9<212>PRT<213>Hepatitis C virus<400>44Ala Thr Pro Pro Gly Ser Ile Thr Val1 5<210>45<211>9<212>PRT<213>Hepatitis C virus<400>45Val Ile Leu Asp Ser Phe Glu Pro Leu1 5<210>46<211>9
<212>PRT<213>Hepatitis C virus<400>46Ile Leu Pro Cys Ser Tyr Thr Thr Leu1 5<210>47<211>9<212>PRT<213>Hepatitis C virus<400>47Phe Trp Ala Lys His Met Trp Asn Phe1 5<210>48<211>9<212>PRT<213>Hepatitis C virus<400>48Ala Leu Tyr Asp Val Val Ser Thr Leu1 5<210>49<211>9<212>PRT<213>Hepatitis C virus<400>49Ala Phe Tyr Gly Val Trp Pro Leu Leu1 5<210>50<211>9<212>PRT<213>Hepatitis C virus<400>50Pro Tyr Ile Glu Gln Gly Met Gln Leu1 5
<210>51<211>9<212>PRT<213>Hepatitis C virus<400>51Thr Pro Pro Ala Val Pro Gln Thr Phe1 5<210>52<211>9<212>PRT<213>Hepatitis C virus<400>52Trp His Tyr Pro Cys Thr Val Asn Phe1 5<210>53<211>9<212>PRT<213>Hepatitis C virus<400>53Gly Ile Leu Pro Phe Phe Met Phe Phe1 5<210>54<211>9<212>PRT<213>Hepatitis C virus<400>54Gly Leu Ile His Leu His Gln Asn Ile1 5<210>55<211>9<212>PRT<213>Hepatitis C virus
<400>55Leu Met Cys Ala Val His Pro Glu Leu1 5<210>56<211>9<212>PRT<213>Hepatitis C virus<400>56Thr Val Leu Ala Asp Phe Lys Thr Trp1 5<210>57<211>9<212>PRT<213>Hepatitis C virus<400>57Ala Tyr Ser Gln Gln Thr Arg Gly Leu1 5<210>58<211>9<212>PRT<213>Hepatitis C virus<400>58Ala Gln Pro Gly Tyr Pro Trp Pro Leu1 5<210>59<211>9<212>PRT<213>Hepatitis C virus<400>59Pro Leu Leu Arg Asp Glu Val Thr Phe1 5<210>60
<211>9<212>PRT<213>Hepatitis C virus<400>60Ile Leu Met Thr His Phe Phe Ser Ile1 5<210>61<211>9<212>PRT<213>Hepatitis C virus<400>61Ser Tyr Thr Trp Thr Gly Ala Leu Ile1 5<210>62<211>9<212>PRT<213>Hepatitis C virus<400>62Ala Thr Pro Pro Gly Ser Val Thr Phe1 5<210>63<211>9<212>PRT<213>Hepatitis C virus<400>63Leu Leu Pro Arg Arg Gly Pro Arg Leu1 5<210>64<211>9<212>PRT<213>Hepatitis C virus<400>64Leu Met Thr His Phe Phe Ser Ile Leu
1 5<210>65<211>9<212>PRT<213>Hepatitis C virus<400>65Leu Leu Lys Arg Leu His Gln Trp Ile1 5<210>66<211>9<212>PRT<213>Hepatitis C virus<400>66Ala Arg Pro Asp Tyr Asn Pro Pro Leu1 5<210>67<211>9<212>PRT<213>Hepatitis C virus<400>67Ala Tyr Tyr Ser Met Val Gly Asn Trp1 5<210>68<211>9<212>PRT<213>Hepatitis C virus<400>68Lys Phe Pro Ala Ala Met Pro Val Trp1 5<210>69<211>9<212>PRT<213>Hepatitis C virus
<400>69Gln Tyr Thr Leu Leu Phe Asn Ile Leu1 5<210>70<211>9<212>PRT<213>Hepatitis C virus<400>70Leu Val Pro Gly Ala Ala Tyr Ala Phe1 5<210>71<211>9<212>PRT<213>Hepatitis C virus<400>71Arg Tyr Ala Pro Ala Cys Lys Pro Leu1 5<210>72<211>9<212>PRT<213>Hepatitis C virus<400>72Phe Leu Ala Arg Leu Ile Trp Trp Leu1 5<210>73<211>9<212>PRT<213>Hepatitis C virus<400>73Ser Gln Leu Asp Leu Ser Gly Trp Phe1 5
<210>74<211>9<212>PRT<213>Hepatitis C virus<400>74Ser Met Leu Thr Asp Pro Ser His Ile1 5<210>75<211>9<212>PRT<213>Hepatitis C virus<400>75Ile Leu Leu Gly Pro Ala Asp Ser Phe1 5<210>76<211>9<212>PRT<213>Hepatitis C virus<400>76Trp Leu Arg Asp Val Trp Asp Trp Ile1 5<210>77<211>9<212>PRT<213>Hepatitis C virus<400>77Tyr Val Val Leu Leu Phe Leu Leu Leu1 5<210>78<211>9<212>PRT<213>Hepatitis C virus<400>78
Leu Asn Pro Ser Val Ala Ala Thr Leu1 5<210>79<211>9<212>PRT<213>Hepatitis C virus<400>79Tyr Val Tyr Asp His Leu Thr Pro Leu1 5<210>80<211>9<212>PRT<213>Hepatitis C virus<400>80His Tyr Arg Asp Val Leu Lys Glu Met1 5<210>81<211>9<212>PRT<213>Hepatitis C virus<400>81Thr Leu Arg Arg His Val Asp Leu Leu1 5<210>82<211>9<212>PRT<213>Hepatitis C virus<400>82Ala Val Ile Pro Asp Arg Glu Val Leu1 5<210>83<211>9<212>PRT
<213>Hepatitis C virus<400>83Phe Leu Ile Ser Gln Leu Phe Thr Phe1 5<210>84<211>9<212>PRT<213>Hepatitis C virus<400>84Val Ile Leu Asp Ser Phe Glu Pro Leu1 5<210>85<211>9<212>PRT<213>Hepatitis C virus<400>85Ile Leu Pro Cys Ser Tyr Thr Thr Leu1 5<210>86<211>9<212>PRT<213>Hepatitis C virus<400>86Gly Ile Leu Pro Phe Phe Met Phe Phe1 5<210>87<211>9<212>PRT<213>Hepatitis C virus<400>87Pro Leu Leu Arg Asp Glu Val Thr Phe1 5
<210>88<211>9<212>PRT<213>Hepatitis C virus<400>88Thr Pro Pro Ala Val Pro Gln Thr Phe1 5<210>89<211>9<212>PRT<213>Hepatitis C virus<400>89Phe Trp Ala Lys His Met Trp Asn Phe1 5<210>90<211>9<212>PRT<213>Hepatitis C virus<400>90Thr Val Leu Ala Asp Phe Lys Thr Trp1 5<210>91<211>9<212>PRT<213>Hepatitis C virus<400>91Ala Leu Tyr Asp Val Val Ser Thr Leu1 5<210>92<211>9<212>PRT<213>Hepatitis C virus<400>92
Trp His Tyr Pro Cys Thr Val Asn Phe1 5<210>93<211>9<212>PRT<213>Hepatitis C virus<400>93Ala Thr Pro Pro Gly Ser Val Thr Phe1 5<210>94<211>9<212>PRT<213>Hepatitis C virus<400>94Gly Leu Ile His Leu His Gln Asn Ile1 5<210>95<211>9<212>PRT<213>Hepatitis C virus<400>95Ala Tyr Ser Gln Gln Thr Arg Gly Leu1 5<210>96<211>9<212>PRT<213>Hepatitis C virus<400>96Ile Leu Met Thr His Phe Phe Ser Ile1 5<210>97<211>9
<212>PRT<213>Hepatitis C virus<400>97Phe Leu Ala Arg Leu Ile Trp Trp Leu1 5<210>98<211>9<212>PRT<213>Hepatitis C virus<400>98Ala Phe Tyr Gly Val Trp Pro Leu Leu1 5<210>99<211>9<212>PRT<213>Hepatitis C virus<400>99Phe Leu Ile Ser Gln Leu Phe Thr Phe1 5<210>100<211>9<212>PRT<213>Hepatitis C virus<400>100Ile Leu Leu Gly Pro Ala Asp Ser Phe1 5<210>101<211>9<212>PRT<213>Hepatitis C virus<400>101Ser Met Leu Thr Asp Pro Ser His Ile1 5
<210>102<211>9<212>PRT<213>Hepatitis C virus<400>102Ala Tyr Tyr Ser Met Val Gly Asn Trp1 5<210>103<211>9<212>PRT<213>Hepatitis C virus<400>103Gln Tyr Thr Leu Leu Phe Asn Ile Leu1 5<210>104<211>9<212>PRT<213>Hepatitis C virus<400>104Leu Leu Lys Arg Leu His Gln Trp Ile1 5<210>105<211>9<212>PRT<213>Hepatitis C virus<400>105Ser Gln Leu Asp Leu Ser Gly Trp Phe1 5<210>106<211>9<212>PRT<213>Hepatitis C virus
<400>106Trp Leu Arg Asp Val Trp Asp Trp Ile1 5<210>107<211>9<212>PRT<213>Hepatitis C virus<400>107Ile Thr Tyr Ser Thr Tyr Gly Lys Phe1 5<210>108<211>9<212>PRT<213>Hepatitis C virus<400>108Ser Tyr Thr Trp Thr Gly Ala Leu Ile1 5<210>109<211>9<212>PRT<213>Hepatitis C virus<400>109Leu Leu Ser Thr Thr Glu Trp Gln Ile1 5<210>110<211>9<212>PRT<213>Heatitis C virus<400>110Arg Tyr Ala Pro Ala Cys Lys Pro Leu1 5<210>111
<211>9<212>PRT<213>Hepatitis C virus<400>111Arg Leu Ile Trp Trp Leu Gln Tyr Phe1 5<210>112<211>9<212>PRT<213>Hepatitis C virus<400>112Val Leu Ala Asp Phe Lys Thr Trp Leu1 5<210>113<211>9<212>PRT<213>Hepatitis C virus<400>113Leu Val Pro Gly Ala Ala Tyr Ala Phe1 5<210>114<211>9<212>PRT<213>Hepatitis C virus<400>114Asp Leu Pro Gln Ile Ile Gln Arg Leu1 5<210>115<211>9<212>PRT<213>Hepatitis C virus<400>115Trp Ile Cys Thr Val Leu Ala Asp Phe
1 5<210>116<211>9<212>PRT<213>Hepatitis C virus<400>116Phe Tyr Gly Val Trp Pro Leu Leu Leu1 5<210>117<211>9<212>PRT<213>Hepatitis C virus<400>117Leu Leu Leu Ser Ile Leu Gly Pro Leu1 5<210>118<211>9<212>PRT<213>Hepatitis C virus<400>118His Tyr Arg Asp Val Leu Lys Glu Met1 5<210>119<211>9<212>PRT<213>Hepatitis C virus<400>119Leu Ile Trp Trp Leu Gln Tyr Phe Ile1 5<210>120<211>9<212>PRT<213>Hepatitis C virus
<400>120Ala Val Ile Pro Asp Arg Glu Val Leu1 5<210>121<211>9<212>PRT<213>Hepatitis C virus<400>121Thr Arg Pro Pro His Gly Asn Trp Phe1 5<210>122<211>9<212>PRT<213>Hepatitis C virus<400>122Lys Phe Pro Ala Ala Met Pro Val Trp1 5<210>123<211>9<212>PRT<213>Hepatitis C virus<400>123Val Phe Pro Asp Leu Gly Val Arg Val1 5<210>124<211>9<212>PRT<213>Hepatitis C virus<400>124Lys Leu Leu Pro Arg Leu Pro Gly Val1 5
<210>125<211>9<212>PRT<213>Hepatitis C virus<400>125Asp Met Pro Ser Thr Glu Asp Leu Val1 5<210>126<211>9<212>PRT<213>Hepatitis C virus<400>126Tyr Leu Tyr Gly Ile Gly Ser Ala Val1 5<210>127<211>9<212>PRT<213>Hepatitis C virus<400>127Tyr Leu Asn Thr Pro Gly Leu Pro Val1 5<210>128<211>9<212>PRT<213>Hepatitis C virus<400>128Cys Leu Leu Leu Leu Ser Val Gly Val1 5<210>129<211>9<212>PRT<213>Hepatitis C virus<400>129
Leu Leu Leu Ser Val Gly Val Gly Ile1 5<210>130<211>9<212>PRT<213>Hepatitis C virus<400>130Leu Leu Cys Pro Ser Gly His Val Val1 5<210>131<211>9<212>PRT<213>Hepatitis C virus<400>131Ala Ile Leu Ser Pro Gly Ala Leu Val1 5<210>132<211>9<212>PRT<213>Hepatitis C virus<400>132Ser Leu Ile Arg Val Pro Tyr Phe Val1 5<210>133<211>9<212>PRT<213>Hepatitis C virus<400>133Asp Val Trp Asp Trp Ile Cys Thr Val1 5<210>134<211>9<212>PRT
<213>Heatitis C virus<400>134Val Ile Pro Ala Ser Gly Asp Val Val1 5<210>135<211>9<212>PRT<213>Hepatitis C virus<400>135Arg Ala Leu Ala His Gly Val Arg Val1 5<210>136<211>9<212>PRT<213>Hepatitis C virus<400>136Leu Ser Asp Gly Ser Trp Ser Thr Val1 5<210>137<211>9<212>PRT<213>Hepatitis C virus<400>137Lys Leu Gln Asp Cys Thr Met Leu Val1 5<210>138<211>9<212>PRT<213>Hepatitis C virus<400>138Tyr Cys Leu Thr Thr Gly Ser Val Val1 5
<210>139<211>9<212>PRT<213>Hepatitis C virus<400>139Ser Met Leu Thr Asp Pro Ser His Ile1 5<210>140<211>9<212>PRT<213>Hepatitis C virus<400>140Ala Ala Phe Cys Ser Ala Met Tyr Val1 5<210>141<211>9<212>PRT<213>Hepatitis C virus<400>141Tyr Ser Pro Gly Glu Ile Asn Arg Val1 5<210>142<211>9<212>PRT<213>Hepatitis C virus<400>142Tyr Thr Asn Val Asp Gln Asp Leu Val1 5<210>143<211>9<212>PRT<213>Hepatitis C virus<400>143
Leu Arg Asp Glu Val Thr Phe Gln Val1 5<210>144<211>9<212>PRT<213>Hepatitis C virus<400>144Leu Ala Ala Leu Thr Gly Thr Tyr Val1 5<210>145<211>9<212>PRT<213>Hepatitis C virus<400>145Cys Glu Pro Glu Pro Asp Val Thr Val1 5<210>146<211>9<212>PRT<213>Hepatitis C virus<400>146Cys Met Ser Ala Asp Leu Glu Val Val1 5<210>147<211>9<212>PRT<213>Hepatitis C virus<400>147Val Phe Pro Asp Leu Gly Val Arg Val1 5<210>148<211>9
<212>PRT<213>Hepatitis C virus<400>148Tyr Cys Phe Thr Pro Ser Pro Val Val1 5<210>149<211>9<212>PRT<213>Hepatitis C virus<400>149Val Leu Gln Ala Ser Leu Ile Arg Val1 5<210>150<211>9<212>PRT<213>Hepatitis C virus<400>150Lys Gln Ala Glu Ala Ala Ala Pro Val1 5<210>151<211>9<212>PRT<213>Hepatitis C virus<400>151Leu Leu Leu Ala Leu Pro Pro Arg Ala1 5<210>152<211>9<212>PRT<213>Hepatitis C virus<400>152Val Leu As Asp His Tyr Arg As Val1 5
<210>153<211>9<212>PRT<213>Hepatitis C virus<400>153Phe Ser Pro Arg Arg His Glu Thr Val1 5<210>154<211>9<212>PRT<213>Heatitis C virus<400>154Ser Val Ile Asp Cys Asn Thr Cys Val1 5<210>155<211>9<212>PRT<213>Heatitis C virus<400>155Gly Leu Ile Arg Ala Cys Thr Leu Val1 5<210>156<211>9<212>PRT<213>Hepatitis C virus<400>156Thr Val Asn Phe Thr Ile Phe Lys Val1 5<210>157<211>9<212>PRT<213>Hepatitis C virus
<400>157Glu Met Gly Gly Asn Ile Thr Arg Val1 5<210>158<211>9<212>PRT<213>Hepatitis C virus<400>158Pro Leu Leu Arg His His Asn Met Val1 5<210>159<211>9<212>PRT<213>Hepatitis C virus<400>159Gln Leu Asp Leu Ser Gly Trp Phe Val1 5<210>160<211>9<212>PRT<213>Hepatitis C virus<400>160Thr Leu Ala Ala Arg Asn Ala Ser Val1 5<210>161<211>9<212>PRT<213>Hepatitis C virus<400>161Arg Leu Gly Ala Val Gln Asn Glu Val1 5<210>162
<211>9<212>PRT<213>Hepatitis C virus<400>162Val Ile Leu Asp Ser Phe Glu Pro Leu1 5<210>163<211>9<212>PRT<213>Hepatitis C virus<400>163Ala Ala Leu Glu Asn Leu Val Val Leu1 5<210>164<211>9<212>PRT<213>Hepatitis C virus<400>164Leu Leu Glu Asp Thr Asp Thr Pro Ile1 5<210>165<211>9<212>PRT<213>Hepatitis C virus<400>165Val Val Thr Ser Thr Trp Val Leu Val1 5<210>166<211>9<212>PRT<213>Hepatitis C virus<400>166Phe Ser Leu Asp Pro Thr Phe Thr Ile
1 5<210>167<211>9<212>PRT<213>Hepatitis C virus<400>167Thr Ile Pro Ala Ser Ala Tyr Glu Val1 5<210>168<211>9<212>PRT<213>Hepatitis C virus<400>168Asp Leu Leu Glu Asp Thr Asp Thr Pro1 5<210>169<211>9<212>PRT<213>Hepatitis C virus<400>169Leu Leu Leu Ser Ile Leu Gly Pro Leu1 5<210>170<211>9<212>PRT<213>Hepatitis C virus<400>170Val Leu Ala Asp Phe Lys Thr Trp Leu1 5<210>171<211>9<212>PRT<213>Hepatitis C virus
<400>171Ser Ile Leu Gly Ile Gly Thr Val Leu1 5<210>172<211>9<212>PRT<213>Hepatitis C virus<400>172Ala Gly Asp Asn Phe Pro Tyr Leu Val1 5<210>173<211>9<212>PRT<213>Hepatitis C virus<400>173Ile Leu Pro Cys Ser Tyr Thr Thr Leu1 5<210>174<211>9<212>PRT<213>Hepatitis C virus<400>174Val Ala Ala Glu Glu Tyr Val Glu Val1 5<210>175<211>9<212>PRT<213>Hepatitis C virus<400>175Leu Ala Val Ala Val Glu Pro Val Val1 5
<210>176<211>9<212>PRT<213>Hepatitis C virus<400>176Ala Leu Tyr Asp Val Val Ser Thr Leu1 5<210>177<211>9<212>PRT<213>Hepatitis C virus<400>177Phe Leu Ala Arg Leu Ile Trp Trp Leu1 5<210>178<211>9<212>PRT<213>Hepatitis C virus<400>178Arg Leu Leu Ala Pro Ile Thr Ala Tyr1 5<210>179<211>9<212>PRT<213>Hepatitis C virus<400>179Trp Leu Arg Asp Val Trp Asp Trp Ile1 5<210>180<211>9<212>PRT<213>Hepatitis C virus<400>180
Cys Val Asn Gly Ala Cys Trp Thr Val1 5<210>181<211>9<212>PRT<213>Hepatitis C virus<400>181Tyr Val Tyr Asp His Leu Thr Pro Leu1 5<210>182<211>9<212>PRT<213>Hepatitis C virus<400>182Thr Val Val Leu Thr Glu Ser Thr Val1 5<210>183<211>9<212>PRT<213>Hepatitis C virus<400>183Ala Ala Arg Ala Leu Ala His Gly Val1 5<210>184<211>3010<212>PRT<213>Hepatitis C virus<400>184Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro50 55 60Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr180 185 190Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys Ser195 200 205Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro210 215 220Gly Cys Val Pro Cys Val Arg Glu Ser Asn Phe Ser Arg Cys Trp Val225 230 235 240
Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser Ser Ile Pro Thr Thr245 250 255Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Leu Cys260 265 270Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser275 280 285Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln Asp Cys290 295 300Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp305 310 315 320Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln325 330 335Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His340 345 350Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp355 360 365Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp Gly His370 375 380Thr His Val Thr Gly Gly Arg Val Ala Ser Ser Thr Gln Ser Leu Val385 390 395 400Ser Trp Leu Ser Gln Gly Pro Ser Gln Lys Ile Gln Leu Val Asn Thr405 410 415Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser420 425 430Leu Gln Thr Gly Phe Ile Ala Ala Leu Phe Tyr Ala His Arg Phe Asn
435 440 445Ala Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Asp Glu450 455 460Phe Ala Gln Gly Trp Gly Pro Ile Thr His Asp Met Pro Glu Ser Ser465 470475 480Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile485 490 495Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser500 505 510Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr Tyr Ser515 520 525Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Ser Asn Thr Arg Pro530 535 540Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe545 550 555 560Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Val Gly Asn565 570 575Asn Thr Leu Val Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala580 585 590Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met595 600 605Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe610 615 620Thr Val Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu625 630 635 640
Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp645 650 655Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp660 665 670Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly675 680 685Leu Ile His Leu His Arg Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly690 695 700Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp Glu Tyr Ile Leu705 710 715 720Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp725 730 735Met Met Leu Leu Ile Ala Gln Ala Glu Ala Thr Leu Glu Asn Leu Val740 745 750Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Leu Leu Ser Phe755 760 765Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro770 775 780Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu785 790 795 800Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala805 810 815Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser820 825 830Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr835 840 845
Phe Ile Thr Arg Ala Glu Ala His Leu Gln Val Trp Val Pro Pro Leu850 855 860Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Thr Cys Ala Val865 870 875 880His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ala Ile Leu885 890 895Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe900 905 910Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Val915 920 925Ala Gly Gly His Tyr Val Gln Met Ala Phe Met Lys Leu Ala Ala Leu930 935 940Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Arg Asp Trp Ala945 950 955 960His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe965 970 975Ser Asp Met Glu Thr Lys Leu Ile Thr Trp Gly Ala Asp Thr Ala Ala980 985 990Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg Gly Lys995 1000 1005Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Gly Glu Gln Gly Trp1010 1015 1020Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly1025 1030 1035Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn1040 1045 1050
Gln Val Asp Gly Glu Val Gln Val Leu Ser Thr Ala Thr Gln Ser1055 1060 1065Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His1070 1075 1080Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr1085 1090 1095Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala1100 1105 1110Pro Pro Gly Ala Arg Ser Met Thr Pro Cys Thr Cys Gly Ser Ser1115 1120 1125Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Val Pro Val Arg1130 1135 1140Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile1145 1150 1155Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser1160 1165 1170Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly1175 1180 1185Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu Thr1190 1195 1200Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala1205 1210 1215Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly1220 1225 1230Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly
1235 1240 1245Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly1250 1255 1260Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Glu Pro Asn Ile1265 1270 1275Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Gly Pro Ile Thr Tyr1280 1285 1290Ser Thr Tyr Cys Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly1295 1300 1305Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser1310 1315 1320Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr1325 1330 1335Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly1340 1345 1350Ser Ile Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser1355 1360 1365Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu1370 1375 1380Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys1385 1390 1395Lys Cys Asp Glu Leu Ala Ala Lys Leu Thr Gly Leu Gly Leu Asn1400 1405 1410Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr1415 1420 1425
Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly1430 1435 1440Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val1445 1450 1455Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu1460 1465 1470Thr Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Ala Gln Arg Arg1475 1480 1485Gly Arg Thr Gly Arg Gly Arg Ser Gly Ile Tyr Arg Phe Val Thr1490 1495 1500Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys1505 1510 1515Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala1520 1525 1530Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu1535 1540 1545Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr1550 1555 1560Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln1565 1570 1575Ala Gly Asp Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val1580 1585 1590Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp1595 1600 1605Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro1610 1615 1620
Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr1625 1630 1635His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu1640 1645 1650Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala1655 1660 1665Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val1670 1675 1680Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg1685 1690 1695Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser1700 1705 1710His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe1715 1720 1725Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala1730 1735 1740Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu1745 1750 1755Val Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln1760 1765 1770Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala1775 1780 1785Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr1790 1795 1800Gln Asn Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala1805 1810 1815
Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly1820 1825 1830Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu1835 1840 1845Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu1850 1855 1860Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp1865 1870 1875Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val1880 1885 1890Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro1895 1900 1905Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala1910 1915 1920Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser1925 1930 1935Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Ser Leu Thr Ile1940 1945 1950Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp Cys1955 1960 1965Ser Thr Pro Cys Ser Gly Ser Trp Leu Lys Asp Val Trp Asp Trp1970 1975 1980Ile Cys Thr Val Leu Ser Asp Phe Lys Thr Trp Leu Gln Ser Lys1985 1990 1995Leu Leu Pro Arg Leu Pro Gly Leu Pro Phe Leu Ser Cys Gln Arg
2000 2005 2010Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Gln Thr Thr2015 2020 2025Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser2030 2035 2040Met Arg Ile Val Gly Pro Lys Thr Cys Ser Asn Thr Trp His Gly2045 2050 2055Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser2060 2065 2070Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala Ala Glu2075 2080 2085Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr2090 2095 2100Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val Pro Ala2105 2110 2115Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His Arg Tyr2120 2125 2130Ala Pro Val Cys Lys Pro Leu Leu Arg Glu Glu Val Val Phe Gln2135 2140 2145Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu2150 2155 2160Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro2165 2170 2175Ser His Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly2180 2185 2190
Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala2195 2200 2205Pro Ser Leu Lys Ala Thr Cys Thr Thr His His Asp Ser Pro Asp2210 2215 2220Ala Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly2225 2230 2235Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu2240 2245 2250Asp Ser Phe Asp Pro Ile Arg Ala Val Glu Asp Glu Arg Glu Ile2255 2260 2265Ser Val Pro Ala Glu Ile Leu Arg Lys Pro Arg Lys Phe Pro Pro2270 2275 2280Ala Leu Pro Ile Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu2285 2290 2295Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val His Gly2300 2305 2310Cys Pro Leu Pro Ser Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg2315 2320 2325Arg Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val Ser Ser Ala2330 2335 2340Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly Ser Ser2345 2350 2355Ala Val Asp Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln Ala Ser2360 2365 2370Asp Asp Gly Asp Lys Gly Ser Asp Val Glu Ser Tyr Ser Ser Met2375 2380 2385
Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly2390 2395 2400Ser Trp Ser Thr Val Ser Gly Glu Ala Gly Glu Asp Val Val Cys2405 2410 2415Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys2420 2425 2430Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser2435 2440 2445Leu Leu Arg His His Ser Met Val Tyr Ser Thr Thr Ser Arg Ser2450 2455 2460Ala Ser Leu Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val2465 2470 2475Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys2480 2485 2490Ala Ser Thr Val Lys Ala Arg Leu Leu Ser Ile Glu Glu Ala Cys2495 2500 2505Lys Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly2510 2515 2520Ala Lys Asp Val Arg Ser Leu Ser Ser Arg Ala Val Asn His Ile2525 2530 2535Arg Ser Val Trp Glu Asp Leu Leu Glu Asp Thr Glu Thr Pro Ile2540 2545 2550Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro2555 2560 2565Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp2570 2575 2580
Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val2585 2590 2595Ser Thr Leu Pro Gln Ala Val Met Gly Pro Ser Tyr Gly Phe Gln2600 2605 2610Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Thr Trp Lys2615 2620 2625Ser Lys Lys Cys Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe2630 2635 2640Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Thr Glu Glu Ser Ile2645 2650 2655Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Arg2660 2665 2670Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn Ser2675 2680 2685Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val2690 2695 2700Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala2705 2710 2715Thr Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu2720 2725 2730Val Asn Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr2735 2740 2745Gln Glu Asp Ala Ala Ala Leu Arg Ala Phe Thr Glu Ala Met Thr2750 2755 2760Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp
2765 2770 2775Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His2780 2785 2790Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr2795 2800 2805Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Val Arg His Thr Pro2810 2815 2820Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu2825 2830 2835Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu2840 2845 2850Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly2855 2860 2865Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Glu2870 2875 2880Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro2885 2890 2895Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val2900 2905 2910Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala29152920 2925Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr2930 2935 2940Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Ile2945 2950 2955
Pro Ala Ala Ser Gln Leu Asp Leu Ser Gly Trp Phe Val Ala Gly2960 2965 2970Tyr Ash Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro2975 2980 2985Arg Trp Phe Met Leu Cys Leu Leu Leu Leu Ser Val Gly Val Gly2990 2995 3000Ile Tyr Leu Leu Pro Asn Arg3005 3010<210>185<211>3010<212>PRT<213>Hepatitis C virus<400>185Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro50 55 60Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp Pro100 105 110
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu130 135 140Gly Gly Ala AlaArg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr180 185 190Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys Ser195 200 205Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile Met His Ala Pro210 215 220Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val225 230 235 240Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr245 250 255Thr Leu Arg Arg His Val Asp Leu Leu Val Gly Thr Ala Ala Phe Cys260 265 270Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Ile Ser275 280 285Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys290 295 300Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp305 310 315 320
Asp Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ser Gln325 330 335Leu Leu Arg Ile Pro Gln Ala Val Met Asp Met Val Ala Gly Ala His340 345 350Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp355 360 365Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp Gly His370 375 380Thr Arg Val Thr Gly Gly Val Gln Gly His Val Thr Ser Thr Leu Thr385 390 395 400Ser Leu Phe Arg Pro Gly Ala Ser Gln Lys Ile Gln Leu Val Asn Thr405 410 415Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser420 425 430Leu Lys Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Lys Phe Asn435 440 445Ala Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Ser Ile Asp Lys450 455 460Phe Asp Gln Gly Trp Gly Pro Ile Thr Tyr Ala Gln Pro Asp Asn Ser465 470 475 480Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Gln Cys Gly Ile485 490 495Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser500 505 510Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr Tyr Asn515 520 525
Trp Gly Asp Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro530 535 540Pro His Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe545 550 555 560Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Arg Gly Val Gly Asn565 570 575Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Asp Ala580 585 590Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu595 600 605Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe610 615 620Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu625 630 635 640Asp Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp645 650 655Arg Asp Arg Ala Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp660 665 670Gln Ile Leu Pro Cys Ser Tyr Thr Thr Leu Pro Ala Leu Ser Thr Gly675 680 685Leu Ile His Leu His Gln Asn Ile Val Asp Ile Gln Tyr Leu Tyr Gly690 695 700Ile Gly Ser Ala Val Val Ser Ile Ala Ile Lys Trp Glu Tyr Val Val705 710 715 720Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp
725 730 735Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val740 745 750Val Leu Asn Ala Ala Ser Val Val Gly Ala His Gly Met Leu Pro Phe755 760 765Phe Met Phe Phe Cys Ala Ala Trp Tyr Met Lys Gly Arg Leu Val Pro770 775 780Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu785 790 795 800Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Val Ala805 810 815Ser Cys Gly Gly Gly ValPhe Val Gly Leu Ala Leu Leu Thr Leu Ser820825 830Pro Tyr Cys Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr835 840 845Phe Ile Thr Lys Ala Glu Ala His Leu Gln Val Ser Leu Pro Pro Leu850 855 860Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val865 870 875 880His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ser Ile Leu885 890 895Gly Pro Leu Met Val Leu Gln Ala Ser Leu Ile Arg Val Pro Tyr Phe900 905 910Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala915 920 925
Ala Gly Gly His Tyr Val Gln Met Ala Phe Val Lys Leu Ala Ala Leu930 935 940Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Gln Asp Trp Ala945 950 955 960His Val Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe965 970 975Ser Ala Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala980 985 990Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg Gly Lys995 1000 1005Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Glu Gly Gln Gly Trp1010 1015 1020Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly1025 1030 1035Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn1040 1045 1050Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Lys Gln Ser1055 1060 1065Phe Leu Ala Thr Cys Val Asn Gly Ala Cys Trp Thr Val Phe His1070 1075 1080Gly Ala Gly Ser Lys Thr Leu Ala Ala Ala Lys Gly Pro Ile Thr1085 1090 1095Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala1100 1105 1110Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser1115 1120 1125
Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg1130 1135 1140Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile1145 1150 1155Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser1160 1165 1170Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly1175 1180 1185Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu Thr1190 1195 1200Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr Pro Pro Ala1205 1210 1215Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly1220 1225 1230Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly1235 1240 1245Tyr Mer Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly1250 1255 1260Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile1265 1270 1275Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr1280 1285 1290Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly1295 1300 1305Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser1310 1315 1320
Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr1325 1330 1335Val Gly Ala Arg Phe Val Val Leu Ala Thr Ala Thr Pro Pro Gly1340 1345 1350Ser Ile Thr Phe Pro His Pro Asn Ile Glu Glu Val Pro Leu Ala1355 1360 1365Asn Thr Gly Glu Ile Pro Phe Tyr Ala Lys Thr Ile Pro Ile Glu1370 1375 1380Val Ile Arg Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys1385 1390 1395Lys Cys Asp Glu Leu Pro Ala Lys Leu Ser Ala Leu Gly Leu Asn1400 1405 1410Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Ala1415 1420 1425Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly1430 1435 1440Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val1445 1450 1455Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu1460 1465 1470Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg1475 1480 1485Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr1490 1495 1500Pro Gly Glu Arg Pro Ser Ala Met Phe Asp Ser Ser Val Leu Cys
1505 1510 1515Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala1520 1525 1530Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu1535 1540 1545Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr1550 1555 1560Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln1565 1570 1575Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val1580 1585 1590Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Gln Met Trp1595 1600 1605Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro1610 1615 1620Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr1625 1630 1635His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu1640 1645 1650Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala1655 1660 1665Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val1670 1675 1680Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg1685 1690 1695
Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser1700 1705 1710His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe1715 1720 1725Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala1730 1735 1740Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu1745 1750 1755Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln1760 1765 1770Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala1775 1780 1785Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Ala Thr1790 1795 1800Gln Tyr Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala1805 1810 1815Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly1820 1825 1830Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu1835 1840 1845Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu1850 1855 1860Val Ala Phe Lys Val Met Ser Gly Asp Met Pro Ser Thr Glu Asp1865 1870 1875Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val1880 1885 1890
Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro1895 1900 1905Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala1910 1915 1920Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser1925 1930 1935Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Asn Leu Thr Ile1940 1945 1950Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp Cys1955 1960 1965Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp1970 1975 1980Ile Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser Lys1985 1990 1995Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg2000 2005 2010Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr2015 2020 2025Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser2030 2035 2040Met Arg Ile Val Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly2045 2050 2055Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser2060 2065 2070Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala Ala Glu2075 2080 2085
Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr2090 2095 2100Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val Pro Ala2105 2110 2115Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Tyr2120 2125 2130Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp Glu Val Thr Phe Gln2135 2140 2145Val Gly Leu Asn Gln Tyr Thr Val Gly Ser Gln Leu Pro Cys Glu2150 2155 2160Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp Pro2165 2170 2175Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala Arg Gly2180 2185 2190Ser Pro Pro Ser Leu Ala Gly Ser Ser Ala Ser Gln Leu Ser Ala2195 2200 2205Leu Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro Asp2210 2215 2220Thr Asp Leu lle Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly2225 2230 2235Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu2240 2245 2250Asp Ser Phe Glu Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val2255 2260 2265Ser Ala Ala Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala
2270 2275 2280Ala Met Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu2285 2290 2295Glu Ser Trp Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly2300 2305 2310Cys Pro Leu Pro Pro Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg2315 2320 2325Arg Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val Ser Ser Ala2330 2335 2340Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Gly Ser Gly Ser Ser2345 2350 2355Ala Val Asp Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln Ala Ser2360 2365 2370Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu Ser Tyr Ser Ser Met2375 2380 2385Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly2390 2395 2400Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys2405 2410 2415Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys2420 2425 2430Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Pro2435 2440 2445Leu Leu Arg His His Asn Met Val Tyr Ser Thr Thr Ser Arg Ser2450 2455 2460
Ala Ser Leu Arg Gln Lys Lys Val Thr Phe Asp Arg Met Gln Val2465 2470 2475Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys2480 2485 2490Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys2495 2500 2505Lys Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly2510 2515 2520Ala Lys Asp Val Arg Ser Leu Ser Ser Arg Ala Val Asn His Ile2525 2530 2535Arg Ser Val Trp Lys Asp Leu Leu Glu Asp Thr Asp Thr Pro Ile2540 2545 2550Gln Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro2555 2560 2565Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp2570 2575 2580Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val2585 2590 2595Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly Phe Gln2600 2605 2610Tyr Ser Pro Lys Gln Arg Val Glu Phe Leu Val Asn Thr Trp Lys2615 2620 2625Ala Lys Lys Cys Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe2630 2635 2640Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile2645 2650 2655
Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Arg2660 2665 2670Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Met Thr Asn Ser2675 2680 2685Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val2690 2695 2700Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala2705 2710 2715Ala Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu2720 2725 2730Val Cys Gly Asp Asp Leu Val Val Ile Cys Asp Ser Ala Gly Thr2735 2740 2745Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala Met Thr2750 2755 2760Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp2765 2770 2775Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His2780 2785 2790Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr2795 2800 2805Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro2810 2815 2820Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu2825 2830 2835Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu2840 2845 2850
Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly2855 2860 2865Ala Thr Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln2870 2875 2880Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro2885 2890 2895Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val2900 2905 2910Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala2915 2920 2925Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr2930 2935 2940Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Ile2945 2950 2955Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp Phe Val Ala Gly2960 2965 2970Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro2975 2980 2985Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly2990 2995 3000Ile Tyr Leu Leu Pro Asn Arg3005 3010<210>186<211>3010<212>PRT<213>Hepatitis C virus
<400>186Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro50 55 60Ile Pro Lys Ala Arg His Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr180 185 190Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys Ser
195 200 205Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile Met His Ala Pro210 215 220Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val225 230 235 240Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr245 250 255Thr Leu Arg Arg His Val Asp Leu Leu Val Gly Thr Ala Ala Phe Cys260 265 270Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Ile Ser275 280 285Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys290 295 300Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp305 310 315 320Asp Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ser Gln325 330 335Leu Leu Arg Ile Pro Gln Ala Val Met Asp Met Val Ala Gly Ala His340 345 350Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp355 360 365Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp Gly His370 375 380Thr Arg Val Thr Gly Gly Val Gln Gly His Val Thr Ser Thr Leu Thr385 390 395 400
Ser Leu Phe Arg Pro Gly Ala Ser Gln Lys Ile Gln Leu Val Asn Thr405 410 415Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser420 425 430Leu Lys Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Lys Phe Asn435 440 445Ala Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Ser Ile Asp Lys450 455 460Phe Asp Gln Gly Trp Gly Pro Ile Thr Tyr Ala Gln Pro Asp Asn Ser465 470 475 480Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Gln Cys Gly Ile485 490 495Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser500 505 510Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr Tyr Asn515 520 525Trp Gly Asp Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr Arg Pro530 535 540Pro His Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe545 550 555 560Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Arg Gly Val Gly Asn565 570 575Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Asp Ala580 585 590Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu595 600 605
Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe610 615 620Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu625 630 635 640Asp Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp645 650 655Arg Asp Arg Ala Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp660 665 670Gln Ile Leu Pro Cys Ser Tyr Thr Thr Leu Pro Ala Leu Ser Thr Gly675 680 685Leu Ile His Leu His Gln Asn Ile Val Asp Ile Gln Tyr Leu Tyr Gly690 695 700Ile Gly Ser Ala Val Val Ser Ile Ala Ile Lys Trp Glu Tyr Val Val705 710 715 720Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp725 730 735Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val740 745 750Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Pro Phe755 760 765Phe Met Phe Phe Cys Ala Ala Trp Tyr Met Lys Gly Arg Leu Val Pro770 775 780Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu785 790 795 800Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala805 810 815
Ser Cys Gly Gly Gly Val Phe Val Gly Leu Ala Leu Leu Thr Leu Ser820 825 830Pro Tyr Cys Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr835 840 845Phe Ile Thr Lys Ala Glu Ala His Leu Gln Val Trp Val Pro Pro Leu850 855 860Asn Val Arg Ala Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val865 870 875 880His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ser Ile Leu885 890 895Gly Pro Leu Met Val Leu Gln Ala Ser Leu Ile Arg Val Pro Tyr Phe900 905 910Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Thr Leu Val Arg Lys Ala915 920 925Ala Gly Gly His Tyr Val Gln Met Ala Phe Val Lys Leu Ala Ala Leu930 935 940Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Gln Asp Trp Ala945 950 955 960His Val Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe965 970 975Ser Ala Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala980 985 990Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg Gly Lys995 1000 1005Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Glu Gly Gln Gly Trp
1010 1015 1020Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly1025 1030 1035Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn1040 1045 1050Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser1055 1060 1065Phe Leu Ala Thr Cys Val Asn Gly Ala Cys Trp Thr Val Phe His1070 1075 1080GIy Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr1085 1090 1095Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala1100 1105 1110Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser1115 1120 1125Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg1130 1135 1140Arg Arg Gly Asp Thr Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile1145 1150 1155Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser1160 1165 1170Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly1175 1180 1185Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu Thr1190 1195 1200
Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr Pro Pro Ala1205 1210 1215Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly1220 1225 1230Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly1235 1240 1245Tyr Met Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly1250 1255 1260Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile1265 1270 1275Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr1280 1285 1290Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly1295 1300 1305Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser1310 1315 1320Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr1325 1330 1335Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly1340 1345 1350Ser Val Thr Phe Pro His Pro Asn Ile Glu Glu Val Ala Leu Gly1355 1360 1365Asn Thr Gly Gln Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu1370 1375 1380Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys1385 1390 1395
Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Pro Leu Gly Leu Asn1400 1405 1410Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Ala1415 1420 1425Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly1430 1435 1440Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val1445 1450 1455Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu1460 1465 1470Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg1475 1480 1485Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr1490 1495 1500Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys1505 1510 1515Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala1520 1525 1530Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu1535 1540 1545Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr1550 1555 1560Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln1565 1570 1575Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val1580 1585 1590
Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Gln Met Trp1595 1600 1605Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro1610 1615 1620Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr1625 1630 1635His Pro Ile Thr Lys Phe Ile Met Ala Cys Met Ser Ala Asp Leu1640 1645 1650Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala1655 1660 1665Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val1670 1675 1680Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg1685 1690 1695Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser1700 1705 1710His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe1715 1720 1725Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala1730 1735 1740Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu1745 1750 1755Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln1760 1765 1770Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala
1775 1780 1785Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Ala Thr1790 1795 1800Gln Tyr Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala1805 1810 1815Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly1820 1825 1830Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu1835 1840 1845Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu1850 1855 1860Val Ala Phe Lys Val Met Ser Gly Asp Met Pro Ser Thr Glu Asp1865 1870 1875Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val1880 1885 1890Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro1895 1900 1905Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala1910 1915 1920Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser1925 1930 1935Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Asn Leu Thr Ile1940 1945 1950Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp Cys1955 1960 1965
Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp1970 1975 1980Ile Cys Thr Val Leu Ala Asp Phe Lys Thr Trp Leu Gln Ser Lys1985 1990 1995Leu Leu Pro Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg2000 2005 2010Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met Tyr Thr Thr2015 2020 2025Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn Gly Ser2030 2035 2040Met Arg Ile Val Gly Pro Arg Thr Cys Ser Asn Thr Trp His Gly2045 2050 2055Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser2060 2065 2070Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala Ala Glu2075 2080 2085Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr2090 2095 2100Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val Pro Ala2105 2110 2115Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Tyr2120 2125 2130Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp Glu Val Thr Phe Gln2135 2140 2145Val Gly Leu Asn Gln Tyr Thr Val Gly Ser Gln Leu Pro Cys Glu2150 2155 2160
Pro Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp Pro2165 2170 2175Ser His Ile Thr Ala Glu Ala Ala Arg Arg Arg Leu Ala Arg Gly2180 2185 2190Ser Pro Pro Ser Leu Ala Gly Ser Ser Ala Ser Gln Leu Ser Ala2195 2200 2205Pro Ser Leu Lys Ala Thr Cys Thr Thr His His Gly Ala Pro Asp2210 2215 2220Thr Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly2225 2230 2235Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Ile Val Ile Leu2240 2245 2250Asp Ser Phe Glu Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val2255 2260 2265Ser Ala Ala Ala Glu Ile Leu Arg Lys Thr Arg Lys Phe Pro Ala2270 2275 2280Ala Met Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu2285 2290 2295Glu Ser Trp Lys Asn Pro Asp Tyr Val Pro Pro Val Val His Gly2300 2305 2310Cys Pro Leu Pro Pro Thr Lys Ala Pro Pro Ile Pro Pro Pro Arg2315 2320 2325Arg Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val Ser Ser Ala2330 2335 2340Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Gly Ser Gly Ser Ser2345 2350 2355
Ala Val Asp Ser Gly Thr Ala Thr Gly Pro Pro Asp Gln Ala Ser2360 2365 2370Ala Glu Gly Asp Ala Gly Ser Asp Ala Glu Ser Tyr Ser Ser Met2375 2380 2385Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly2390 2395 2400Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys2405 2410 2415Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys2420 2425 2430Ala Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Pro2435 2440 2445Leu Leu Arg His His Asn Met Val Tyr Ser Thr Thr Ser Arg Ser2450 2455 2460Ala Ser Leu Arg Gln Lys Lys Val Thr Phe Asp Arg Met Gln Val2465 2470 2475Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys2480 2485 2490Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys2495 2500 2505Lys Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly2510 2515 2520Ala Lys Asp Val Arg Ser Leu Ser Ser Arg Ala Val Asn His Ile2525 2530 2535Arg Ser Val Trp Lys Asp Leu Leu Glu Asp Thr Asp Thr Pro Ile
2540 2545 2550Gln Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro2555 2560 2565Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp2570 2575 2580Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val2585 2590 2595Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly Phe Gln2600 2605 2610Tyr Ser Pro Lys Gln Arg Val Glu Phe Leu Val Asn Thr Trp Lys2615 2620 2625Ala Lys Lys Cys Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe2630 2635 2640Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile2645 2650 2655Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Arg2660 2665 2670Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Met Thr Asn Ser2675 2680 2685Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val2690 2695 2700Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala2705 2710 2715Ala Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu2720 2725 2730
Val Cys Gly Asp Asp Leu Val Val Ile Cys Asp Ser Ala Gly Thr2735 2740 2745Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala Met Thr2750 2755 2760Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp2765 2770 2775Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His2780 2785 2790Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr2795 2800 2805Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro2810 2815 2820Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu2825 2830 2835Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu2840 2845 2850Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly2855 2860 2865Ala Thr Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln2870 2875 2880Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro2885 2890 2895Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val2900 2905 2910Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala2915 2920 2925
Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr2930 2935 2940Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Ile2945 2950 2955Pro Glu Ala Ser Gln Leu Asp Leu Ser Gly Trp Phe Val Ala Gly2960 2965 2970Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro2975 2980 2985Arg Trp Phe Met Trp Cys Leu Leu Leu Leu Ser Val Gly Val Gly2990 2995 3000Ile Tyr Leu Leu Pro Asn Arg3005 3010
權(quán)利要求
1.與HLA-A型分子結(jié)合的HLA-結(jié)合肽，所述HLA-結(jié)合肽包含至少一種選自SEQ ID NOS1-183的氨基酸序列，以及不少于8個且不多于11個氨基酸殘基。
2.權(quán)利要求1所述的HLA-結(jié)合肽，包含至少一種選自SEQ IDNOS1，2，3，5，8，12，13，14，16，17，18，19，22，23，25，27，34，37，38，40，42，45，48，49，52，53，54，55，56，58，59，60，61，62，63，64，65，67，71，72，74，75，76，84，86，87，90，91，92，93，94，96，97，98，100，101，102，104，106，107，108，109，110，112，123，124，126，127，131，132，133，134，135，136，137，139，141，142，146，147，149，150，152，162，170，173，176，177，和179的氨基酸序列。
3.與HLA-A型分子結(jié)合的HLA-結(jié)合肽，所述HLA-結(jié)合肽包含在權(quán)利要求1或2所述的HLA-結(jié)合肽中所含的所述氨基酸序列的基礎(chǔ)上，通過缺失，替換，或添加一個或兩個氨基酸殘基而形成的氨基酸序列，以及不少于8個且不多于11個氨基酸殘基。
4.權(quán)利要求1-3任一項所述的HLA-結(jié)合肽，其中所述HLA-結(jié)合肽與人HLA-A24型分子結(jié)合。
5.權(quán)利要求1-3任一項所述的HLA-結(jié)合肽，其中所述HLA-結(jié)合肽與人HLA-A2型分子結(jié)合。
6.含有DNA序列的DNA區(qū)段，所述DNA序列編碼權(quán)利要求1-5任一項所述的HLA-結(jié)合肽。
7.含有DNA序列的重組載體，所述DNA序列編碼權(quán)利要求1-5任一項所述的HLA-結(jié)合肽。
8.HLA-結(jié)合肽前體，其在哺乳動物體內(nèi)變成權(quán)利要求1-5任一項所述的HLA-結(jié)合肽。
全文摘要
本發(fā)明提供一種與HLA－A型分子結(jié)合的HLA－結(jié)合肽，所述HLA－結(jié)合肽包含至少一種選自SEQ ID NOS1－183的氨基酸序列，不少于8個且不多于11個氨基酸殘基。通過預(yù)測程序，利用圖1中所述的積極學(xué)習(xí)實驗方法，所有氨基酸序列被預(yù)測具有與人HLA－A型分子的結(jié)合性質(zhì)。
文檔編號C07K14/18GK1954068SQ20058001376
公開日2007年4月25日 申請日期2005年4月14日 優(yōu)先權(quán)日2004年4月30日
發(fā)明者宮川知也, 宇高惠子 申請人:日本電氣株式會社, 國立大學(xué)法人高知大學(xué)