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攜帶乙肝病毒抗原表位的重組嵌合蛋白及其制備的制作方法

文檔序號:604180閱讀:564來源:國知局
專利名稱:攜帶乙肝病毒抗原表位的重組嵌合蛋白及其制備的制作方法
技術領域
本發(fā)明涉及一種重組嵌合蛋白,尤其是一種以輪狀病毒VP6蛋白為載體攜帶乙肝病毒抗原表位的重組嵌合蛋白及其制備,屬于醫(yī)學生物技術領域。
背景技術
輪狀病毒(Rotavirus,簡稱RV)是嬰幼兒腹瀉的主要病原體。世界范圍內每 年大約有60多萬例RV感染患者死亡,其中80%發(fā)生在發(fā)展中國家。RV屬呼腸病毒科(Meoviridae),輪狀病毒屬(Rotavirus)0 RV基因組由11個片段的雙鏈RNA組成,分別編碼6個結構蛋白(VP1-VP4, VP6和VP7)和6個非結構蛋白(NSP1-NSP5/NSP6)。根據(jù)RV組(亞組)抗原蛋白VP6的抗原性的不同,可將至今已發(fā)現(xiàn)的輪狀病毒RV分為A G七個組,A組是嬰幼兒腹瀉的主要病原體。根據(jù)病毒蛋白編碼框(ORF)核苷酸序列同源性差異,可將A組輪狀病毒RV的12個蛋白(基因)分為不同的基因型。根據(jù)RV基因型分類標準,A組輪狀病毒RV的結構蛋白VP6至少可分為16個基因型(11-116型)。VP6蛋白由第6基因片段編碼,含397個氨基酸,分子量約為44. 8KDa。VP6蛋白是輪狀病毒RV中含量最豐富的蛋白,約占病毒總蛋白量的51%。RV內殼中間層由260個致密的VP6蛋白三聚體共780個蛋白分子構成。VP6蛋白具有重要的生物學功能,是病毒基因組轉錄和病毒組裝不可缺少的成分。VP6蛋白也具有重要的免疫學性質。近年進行的VP6蛋白免疫學研究表明,一些抗VP6蛋白的IgA單克隆抗體諸如7D9所產生的IgA具有保護作用,在SCID鼠中可清除其慢性RV病毒感染。一些研究表明VP6蛋白的抗病毒作用可能是通過IgA的分泌途徑而阻礙了病毒的復制循環(huán)過程,或作為一種黏膜的分泌型IgAJf RV感染的上皮細胞排除,從而起到保護作用。研究發(fā)現(xiàn)VP6蛋白分子中至少存在兩個Th細胞抗原表位和一個交叉反應的細胞毒性T淋巴細胞(CTL)表位。有研究表明在使用麥芽糖結合VP6蛋白對小鼠進行黏膜免疫后,IFN- Y被認為是⑶4+T細胞分泌的唯一有效的抗RV的細胞因子。眾多的實驗表明,經VP6蛋白免疫后,在動物體內確實能引起抗RV感染的作用。抗VP6蛋白單克隆抗體具有抑制病毒轉錄活性和保護小鼠免受病毒感染的作用。用重組表達的VP6蛋白免疫小鼠,也可誘導小鼠產生免疫保護作用。VP6蛋白可自我組裝成病毒樣顆粒(VLPs)。由于VLPs沒有感染性,在研究細胞相互作用,開發(fā)診斷制劑和疫苗方面,具有更好的安全性。與單一分子的重組抗原相比,VLPs具有更好的免疫原性,可刺激機體產生體液和細胞免疫反應。由于VLPs具有高密度的呈遞抗原表位,可產生很強的免疫反應,是許多疫苗研制中很有前景的材料。至今所有已用于人體的輪狀病毒RV疫苗都是減毒活疫苗。1997年始用于人體的多價減毒重組活疫苗Rotashield因后來發(fā)現(xiàn)與腸套疊有關聯(lián)而在使用一年后即停止使用。2006年和2008年分別獲準在人群中使用的5價重組減毒活疫苗RotaTeq和單價重組減毒活疫苗Rotarix現(xiàn)也已發(fā)現(xiàn)有豬環(huán)狀病毒DNA污染。Rotarix已在美國人群中停止使用,RotaTeq在人群中尚可正常使用。由于上述用于人類的RV疫苗存在腸套疊和其他病毒核酸污染等風險,且都存在只能降低RV感染引起的死亡率和重癥腹瀉,不能完全保護RV感染的不足,在Rotarix停用后,RV感染率似有所提高。乙肝病毒(HeptatitisB Virus,簡稱 HBV)屬嗜肝DNA病毒科(Hepadnaviridae),是引起人類急慢性肝炎、肝硬化及肝癌的重要病原。我國現(xiàn)有慢性HBV感染者9300萬人。至今已有的能夠有效治療乙肝的藥物都存在副作用大的問題,因此,疫苗免疫仍然是主要的控制措施。乙肝難于治療和預防的重要原因,是因為HBV基因組結構復雜,變異很大。根據(jù)HBV基因核苷酸(氨基酸)序列同源性差異,至今發(fā)現(xiàn)的HBV株至少可以分為10個基因型(基因型A-J)和數(shù)十個基因亞型。因此對HBV蛋白免疫學性質進行深入研究,改進疫苗免疫保護效果,仍有重要的理論意義和使用價值。研制重組抗原表位亞單位疫苗是一個重要的方向。HBV抗原表位在表面抗原蛋白PreSl,PreS2, S及核心蛋白C上均有分布,在尚無理想的治療RV和HBV感染藥物,以及RV和HBV疫苗尚不十分理想的今天,選擇和鑒定這些蛋白上的抗原表位,研制更加安全有效的RV和HBV疫苗,特別是研制可同時保護RV/HBV感染的聯(lián)合疫苗具有重要的經濟和社會價值。

發(fā)明內容
本發(fā)明的目的在于提供一種攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白及制備,以用作RV/HBV重組嵌合疫苗及試劑。本發(fā)明的目的還在于提供一種攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白及制備,以用作RV/HBV重組嵌合疫苗及試劑。本發(fā)明的目的又在于提供一種攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白及制備,以用作RV/HBV重組嵌合疫苗及試劑。本發(fā)明的目的在于提供一種攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白及制備,以用作RV/HBV重組嵌合疫苗及試劑。本發(fā)明的目的在于提供一種攜帶有乙肝病毒SI [93]抗原表位的重組嵌合蛋白及制備,以用作RV/HBV重組嵌合疫苗及試劑。本發(fā)明的目的在于提供一種攜帶有乙肝病毒C117抗原表位的重組嵌合蛋白及制備,以用作RV/HBV重組嵌合疫苗及試劑。本發(fā)明的另一目的在于提供一種同時攜帶乙肝病毒382,(19,5125,52[8],51[93]和C117抗原表位的重組嵌合蛋白及制備,以用作RV/HBV重組嵌合疫苗及試劑。本發(fā)明提供的攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白的氨基酸序列為 Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He Val
Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys Leu Ser Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Phe Leu Phe lieLeu Leu Leu Cys Leu Glu Leu Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu lie GlnVal Ala Gly Phe Asp Tyr Ser Asn He Gln Gln Phe Glu His lie Val Gln Leu Arg ArgVal Leu Thr Thr Ala Thr He Thr Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro ArgVal He Asn Ser Ala Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val lie Leu Arg Pro AsnAsn Val Glu Val Glu Phe Leu Leu Asn Gly Gln He lie Asn Thr Tyr Gln Ala Pro ArgPhe Gly Thr He Val Ala Arg Asn Phe Asp Thr He Arg Leu Ser Phe Gln Leu Met ArgPro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val Gly Leu Thr Leu ArgHe Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala AsnVal Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly MetAsn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg ValPhe Thr Val Ala Ser lie Arg Ser Met Leu Val Lys0所述抗原表位S82的氨基酸序列為Phe Leu Phe He Leu Leu Leu Cys Leu。本發(fā)明提供的攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白的氨基酸序列為 Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He Val
Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn Ser Phe LeuPro Ser Asp Phe Phe Pro Ser Val Arg Asp Ser Asn He Gln Gln Phe Glu His He ValGln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu Pro Asp Ala Glu Arg PheSer Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val IleLeu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile He Asn Thr TyrGln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp Thr He Arg Leu Ser PheGln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val GlyLeu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu ThrMet Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val PhePro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp AsnLeu Gln Arg Val Phe Thr Val Ala Ser He Arg Ser Met Leu Val Lys0所述抗原表位C19 的氨基酸序列為Ser Phe Leu Pro Ser Asp Phe Phe Pro SerVal Arg Asp。本發(fā)明提供的攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白的氨基酸序列為 Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He Val
Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly lie Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr lie Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn lie Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn lie Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr lie Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Pro AlaGln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Thr Thr TrpTyr Phe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn GlyGln He He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr lie Val Ala Arg Asn Phe AspThr He Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu GluHis His Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala lie Cys Glu Ser Val LeuAla Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala liePro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu lie Thr Asn Tyr SerPro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser lie Arg Ser Met LeuVal Lys0所述抗原表位S125 的氨基酸序列為Thr Thr Pro Ala Gln Gly Asn Ser Met PhePro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly0本發(fā)明提供的攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白的氨基酸序列為 Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He Val
Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn Trp Asn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnHe He Asn Thr Tyr Gln Ala Pro Arg Phe His Gln Ala Leu Gln Asp Pro Arg Val ArgGly Leu Tyr Phe Pro Ala Gly Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp ThrHe Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu HisHis Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu AlaAsp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala lie ProVal Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu lie Thr Asn Tyr Ser ProSer Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser lie Arg Ser Met Leu ValLys o
所述抗原表位S2 [8]的氨基酸序列為Pro Arg Phe His Gln Ala Leu Gln AspPro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly。本發(fā)明提供的攜帶有乙肝病毒Sl[93]抗原表位的重組嵌合蛋白的氨基酸序列為
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnHe He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp ThrHe Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Pro Pro Pro Ala Ser Thr Asn Arg GlnSer Gly Arg Gln Pro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val GlyLeu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu ThrMet Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val PhePro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp AsnLeu Gln Arg Val Phe Thr Val Ala Ser He Arg Ser Met Leu Val Lys0所述抗原表位SI [93]的氨基酸序列為Pro Pro Pro Ala Ser Thr Asn Arg GlnSer Gly Arg Gin。本發(fā)明提供的攜帶有乙肝病毒C117抗原表位的重組嵌合蛋白的氨基酸序列為 Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He Val
Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn lie Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr lie Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val lie Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnHe He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr lie Val Ala Arg Asn Phe Asp ThrHe Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu TyrLeu Val Ser Phe Gly Val Trp He Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala ProHe Leu Glu His His Ala Thr Val Gly Leu Thr Leu Arg lie Glu Ser Ala lie Cys GluSer Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln GluTyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu lie ThrAsn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser lie ArgSer Met Leu Val Lys0 所述抗原表位Cl17 的氨基酸序列為Tyr Leu Val Ser Phe Gly Val Trp He ArgThr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro lie。本發(fā)明提供的同時攜帶乙肝病毒S82,C19,S125,S2[8],SI [93]和C117抗原表位的重組嵌合蛋白的氨基酸序列為
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Phe Leu Phe IleLeu Leu Leu Cys Leu Glu Leu Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu He GlnVal Ala Gly Phe Asp Tyr Ser Asn Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val ArgAsp Ser Asn He Gln Gln Phe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr AlaThr He Thr Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser AlaAsp Gly Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys ProThr Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu ValGlu Phe Leu Leu Asn Gly Gln Ile He Asn Thr Tyr Gln Ala Pro Arg Phe His Gln AlaLeu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Pro Arg Phe Gly Thr IleVal Ala Arg Asn Phe Asp Thr He Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Pro ProPro Ala Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Arg Thr Pro Ser Val Ala Ala LeuGlu Tyr Leu Val Ser Phe Gly Val Trp He Arg Thr Pro Pro Ala Tyr Arg Pro Pro AsnAla Pro He Leu Glu His His Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala IleCys Glu Ser Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val ArgGln Glu Tyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp LeuHe Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Serlie Arg Ser Met Leu Val Lys0本發(fā)明提供的攜 帶有乙肝病毒S82抗原表位的重組嵌合蛋白,攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白,攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白,攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白,攜帶有乙肝病毒Sl[93]抗原表位的重組嵌合蛋白,攜帶有乙肝病毒C117抗原表位的重組嵌合蛋白均通過下列步驟制備
IA、根據(jù)抗原表位氨基酸序列,分別設計下列引物
S82F CTTCCTCTTCATCCTGCTGCTATGCCTGGAGCT
S82R CCAGGCATAGCAGCAGGATGAAGAGGAAGAGCT
C19F CGAATCATTTTTGCCTTCTGACTTCTTTCCTTCCGTCCGGGATTTC19R CGAAATCCCGGACGGAAGGAAAGAAGTCAGAAGGCAAAAATGATTS125F CACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGTACS125R CATCCGTAGGTTTTGTACAGCAACATGAGGGAAACATAGAGTTGCCTTGAGCAGGAGTGGTACS2[8] F CTAGGTTCCACCAAGCTCTGCAAGATCCCAGAGTCAGGGGCCTGTATTTTCCTGCTGGTCS2 [8] R CTAGGACCAGCAGGAAAATACAGGCCCCTGACTCTGGGATCTTGCAGAGCTTGGTGGAACSI[93]F GGCCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCGC
SI[93] R GGCTGCCTTCCTGACTGCCGATTGGTGGAGGCAGGAGGAGGCCGC
C117F TCGAGTATTTGGTCTCTTTCGGAGTGTGGATTCGCACTCCACCAGCTTATAGACCACCAAATGCCCCTATCC
C117R TCGAGGATAGGGGCATTTGGTGGTCTATAAGCTGGTGGAGTGCGAATCCACACTCCGAAAGAGACCAAATAC ;
IB、將步驟IA的各個抗原表位的引物進行如下配對
將S抗原表位S82的兩條人工合成的寡核苷酸即正鏈S82F和負鏈S82R配成引物對S82F/S82R ;
將C抗原表位C19,其氛基酸序列為Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser ValArg Asp的兩條人工合成的寡核苷酸即正鏈(C19F)和負鏈(C19R)配成引物對C19F/C19R;
將S抗原表位S125的兩條人工合成的寡核苷酸即正鏈S125F和負鏈S125R配成引物對 S125F/S125R ;
將PreS2抗原表位S2 [8]的兩條人工合成的寡核苷酸即正鏈S2 [8] F和負鏈S2 [8] R配成引物對 S2[8]F/S2[8]R;
將PreSl抗原表位SI [93]的兩條人工合成的寡核苷酸即正鏈SI [93]F和負鏈SI [93]R 配成引物對 SI [93JF/S1 [93] R ;
將C抗原表位Cl 17的兩條人工合成的寡核苷酸即正鏈C117F和負鏈C117R配成引物對 C117F/C117R。1C、將步驟IB的各引物對分別在下列條件下進行退火,退火混合液中分別含有2μ I的正向引物,2 μ I的反向引物,以及7μ I的PCR緩沖液和59 μ I的水;將退火混合液在65°C水浴反應5分鐘后,放入37°C水浴反應45分鐘,反應體系中加入400 μ I的TE,并與等體積異丙醇充分混勻,室溫放置10分鐘后,4°C, 15,OOOrpm離心15min,沉淀用質量濃度為75%的乙醇漂洗,干燥后,用 ομ I的TE溶解;分別得到在5’端和3’端分別帶Sac I位點,或者BspT104 I位點,或者Kpn I位點,或者Bln I位點,或者Sac II位點,或者XhoI位點的下列六作互補雙鏈S82互補雙鏈;C19互補雙鏈;S125互補雙鏈;S2[8]互補雙鏈;SI [93]互補雙鏈;C117互補雙鏈;
1D、將攜帶有 Sac HliUBspTKM I 位點、Kpn I (ggtacc)位點、Sac II (ccgcgg)位點、Sac II (CCRCRR)位點及Xho I (ctcRaR)位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒PETP6F,在下列條件下分別用Sac I,BspT104 I, Kpn I, Bln I, Sac II7Xho I酶切酶切混合液分別含3μ I的Sac I,BspT104 I,Kpn I,Bln I,Sac II或Xho I內切酶(15U),以及10 μ I的pETP6F DNA, 7 μ I的IOX緩沖液,50 μ I的水;將酶切混合液放于37°C水浴反應3小時,得下列六個酶切質粒片段Sac I酶切質粒片段;BspT104 I酶切質粒片段;Kpn I酶切質粒片段;Β1η I酶切質粒片段;Sac II酶切質粒片段;Xho I酶切質粒片段;用I. 5%的瓊脂糖凝膠電泳分離和純化所得六個帶有不同切口的酶切質粒片段;
IE、將步驟ID所得六個酶切質粒片段分別與步驟IC所得六個互補雙鏈配對如下
Sac I酶切質粒片段/S82互補雙鏈;
BspT104 I酶切質粒片段/C19互補雙鏈;
Kpn I酶切質粒片段/S125互補雙鏈;
Bln I酶切質粒片段/S2[8]互補雙鏈;
Sac II酶切質粒片段/SI [93]互補雙鏈;
Xho I酶切質粒片段/Cl 17互補雙鏈;
將上述配對的各片段分別與T4 DNA連接酶及緩沖液混合后,在18°C連接反應6h,分別得到下列六個重組質粒
在Sac I位點攜帶有S82抗原表位基因的重組質粒pETP6F/S82 ;
在BspT104 I位點攜帶有C19抗原表位基因的重組質粒pETP6F/C19 ;
在Kpn I位點攜帶有S125抗原表位基因的重組質粒pETP6F/S125 ;
在Bln I位點攜帶有S2[8]抗原表位基因的重組質粒pETP6F/S2[8];
在Sac II位點攜帶有SI [93]抗原表位基因的重組質粒pETP6F/Sl [93];
在Xho I位點攜帶有Cl 17抗原表位基因的重組質粒pETP6F/C117 ;
按常規(guī)分別將所得重組質粒經菌體轉化擴增后,進行酶切鑒定和核苷酸序列測定;
IF、將步驟IE 得到的六個重組質粒,即 pETP6F/S82,pETP6F/C19, pETP6F236K/S125,pETP6F268B/S2[8], pETP6F290S/Sl [93],pETP6F298X/C117 在下列條件下分別轉化 BL21(DE3)感受態(tài)細胞10 μ I的重組質粒與100 μ I BL21 (DE3)感受態(tài)細胞混勻,冰上放置30分鐘,42°C放置70秒鐘,加入500 μ I LB培養(yǎng)液后,在37°C培養(yǎng)30分鐘,培養(yǎng)板涂含200 μ g/ml氨芐青霉素的I. 5%瓊脂培養(yǎng)板,培養(yǎng)板于37°C培養(yǎng)14個小時,挑取單個菌落放入含200 μ g/ml氨芐青霉素的LB培養(yǎng)液中培養(yǎng)和表達,得到攜帶有不同抗原表位的下列六個重組蛋白
(1)攜帶乙肝病毒S82抗原表位的重組蛋白rVP6F/S82;
(2)攜帶乙肝病毒C19抗原表位的重組蛋白rVP6F/C19;
(3)攜帶乙肝病毒S125抗原表位的重組蛋白rVP6F/S125;
(4)攜帶乙肝病毒S2[8]抗原表位的重組蛋白rVP6F/S2[8];(5)攜帶乙肝病毒SI[93]抗原表位的重組蛋白rVP6F/Sl[93];
(6)攜帶乙肝病毒C117抗原表位的重組蛋白rVP6F/C117。本發(fā)明提供的同時攜帶乙肝病毒S82,C19,S125,S2[8],S1[93]和C117抗原表位的重組蛋白通過下列步驟制備
2A、同上述IA ;
2B、同上述IB ;
2C、同上述IC ;
2D、同上述ID ;
2E、同上述IE ;
2F、將步驟2E所得重組質粒pETP6F/S82用Bspt 104 I酶切,并與步驟2C所得C19互補雙鏈連接,得到在Sac I和BspT104 I位點攜帶有S82和C19抗原表位基因的重組質粒PETP6F/S82/C19 ;
2G、將步驟2E所得重組質粒pETP6F/S82/C19用Kpn I酶切,并與步驟2C所得S125互補雙鏈連接,得到在Sac I, BspT104 I和Kpn I位點攜帶有S82, C19和S125抗原表位基因的重組質粒 PETP6F/S82/C19/S125 ;
2H、將步驟2E所得重組質粒pETP6F/S82/C19/S125用Bln I酶切,并與步驟2C所得S2[8]互補雙鏈連接,得到在Sac I,BspT104 I, Kpn I和Bln I位點攜帶有S82,C19,S125和S2[8]抗原表位基因的重組質粒pETP6F/S82/C19/S125/S2[8];
21、將步驟2E所得重組質粒pETP6F/S82/C19/S125/S2[8]用Sac II酶切,并與步驟2C所得SI [93]互補雙鏈連接,得到在Sac I, BspT104 I,Kpn I,Bln I和Sac II位點攜帶有 S82,C19,S125,S2[8]和 SI [93]抗原表位基因的重組質粒pETP6F/S82/C19/S125/S2[8]/S1[93];
2J、將步驟2E所得重組質粒pETP6F/S82/C19/S125/S2[8]/Sl[93]用Xho I酶切,并與步驟2C所得C117互補雙鏈連接,得到在Sac I, BspT104 I,Kpn I,Bln I, Sac II和Xho I位點攜帶有對應的S82,C19,S125,S2 [8],SI [93]和Cl 17抗原表位基因的重組質粒pETP6F/VP⑥H ;按常規(guī)將所得重組質粒經菌體轉化擴增后,進行酶切鑒定和核苷酸序列測定;
2K、將步驟2J得到的重組質粒pETP6F/VP⑥H在下列條件下轉化BL21(DE3)感受態(tài)細胞10 μ I的步驟2J所得重組質粒與100 μ I BL21 (DE3)感受態(tài)細胞混勻,冰上放置30分鐘,42 V放置70秒鐘,加入500 μ I LB培養(yǎng)液后,在37°C培養(yǎng)30分鐘。培養(yǎng)物涂含200 μ g/ml氨芐青霉素的I. 5%瓊脂培養(yǎng)板;培養(yǎng)板放37°C培養(yǎng)箱中培養(yǎng)14個小時,挑取單個菌落放入含200 μ g/ml氨芐青霉素的LB培養(yǎng)液中培養(yǎng)和表達,得攜帶乙肝病毒S82,C19,S125,S2[8],S1[93]和Cl 17抗原表位的重組蛋白rVP⑥H。所述步驟ID或2D的攜帶有Sac I位點、BspT104 I位點、Kpn I位點、Bln I位點、Sac II位點及Xho I位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒pETP6F通過下列步驟構建
I)人工設計下列引物對
P174F CATGAGCTCATGGGTACAATGTGG ;
P174R CATGAGCTCATGCGCAGGTTGTGA ;
P197F GATTATTCGAACATACAGCAATTTGAGCAT ;P197R TATGTTCGAATAATCAAATCCAGCAAC ;
P244F GATGGTACCACATGGTATTTTAATCCA ;
P244R ;TGTGGTACCATCAGCTGAATTAATTAC ;
P275F GCACCTAGGTTTGGTACAATTGTA ;
P275R AAACCTAGGTGCCTGATAAGTATTTAT ;
P298F AGACCGCGGACCCCATCAGTCGCA ;
P298R GGTCCGCGGTCTCATCAATTGAAATGA ; P308F GCACTCGAGCATCATGCTACTGTA ;
P308R ATGCTCGAGTGCTGCGACTGATGG ;
以及下列引物
PETL5 ATCATATGGAGGTTCTGTACTC ;
PVP6-3 TAGGATCCTTATCATTTAACAAGCATGCTTCTAAT ;
2)以輪狀病毒TB-Chen株VP6蛋白編碼基因為模板,以下列為引物對,進行PCR擴增 PETL5/P174R 和 P174F/PVP6-3 ;
PETL5/P197R 和 P197F/PVP6-3 ;
PETL5/P244R 和 P244F/PVP6-3 ;
PETL5/P275R 和 P275F/PVP6-3 ;
PETL5/P298R 和 P298F/PVP6-3 ;
PETL5/P308R 和 P308F/PVP6-3 ;
具體是
在PCR反應體系為PCR反應混合液體積為100 μ I,包含30ng模板DNA,I X IO5 pmol的dNTPs, IOX PCR緩沖液 10μ 1,5U Taq DNA聚合酶,60pmol 的引物對PETL5/P174R和P174F/PVP6-3,其余用水補足;PCR反應條件為94°C預變性3 min,94°C變性40s,56°C復性30s,72°C延伸反應lmin,共40個循環(huán),最后72°C延伸10 min ;得到帶有NdeI和Sac I位點序列的PCR擴增片段6174F和帶有Sac I和BamH I位點序列的PCR擴增片段6174R ;用I. 5%的瓊脂糖凝膠電泳分離和純化該擴增片段;將PCR擴增片段6174F和6174R分別用Nde I/Sac I 和 Sac I/BamH I 雙酶切,即
A、6174F酶切片段制備酶切反應體系為3μI的6174F片段,3 μ I的Nde I (15U),3μ I的Sac I (15U),8y I的IOX緩沖液,63 μ I的水;將酶切混合液分別于37°C水浴反應3小時后,得酶切片段,用I. 5%的瓊脂糖凝膠電泳分離和純化該酶切片段;
B、除使用SacI/BamH I雙酶切外,6174R酶切片段制備與6174F酶切片段相同;
C、酶切片段的連接將6174F和6174R酶切片段與T4DNA連接酶及緩沖液混合后,在18°C連接反應6h,得到攜帶有Sac I位點(gagctc)的VP6編碼基因
3)運用步驟2)的PCR擴增、酶切和連接,使用對應的PETL5/P197R和P197F/PVP6-3引物對及內切酶Nde I/BspT104 I和BspT104 I/BamH I,在步驟2)獲得的攜帶有Sac I位點的VP6編碼基因上插入BspT104 I (ttcgaa)位點,得到攜帶有Sac I位點和BspT104 I位點的VP6編碼基因;
4 )用與步驟2 )相同的PCR擴增、酶切和連接,使用對應的PETL5/P244R和P244F/PVP6-3引物對及內切酶Nde I/Kpn I和Kpn I/BamH I,在步驟3)所得攜帶有Sac I位點和BspT104 I位點的VP6編碼基因上插入Kpn I (RRtacc)位點,得到攜帶有Sac I位點、BspT104 I位點及Kpn I (ggtacc)位點的VP6編碼某因
5)用與步驟2)相同的PCR擴增、酶切和連接,使用對應的PETL5/P275R和P275F/PVP6-3引物對及內切酶Nde I/Bln I和Bin I/BamH I,在步驟4)所得攜帶有Sac I位點、BspT104 I位點及Kpn I (ggtacc)位點的VP6編碼基因上插入Bln I (cctagg)位點,得至Ij攜帶有 Sac I 位點、BspT104 I 位點、Kpn I (ggtacc)位點及 Bln I (cctagg)位點的 VP6編碼基因;
6)用與步驟2)相同的PCR擴增、酶切和連接,使用對應的PETL5/P298R和P298F/PVP6-3引物對及內切酶Nde I/Sac II和Sac I I/BamH I,在步驟5)所得攜帶有Sac I位點、BspT104 I位點、Kpn I (ggtacc)位點及Bln I (cctagg)位點的VP6編碼基因上插入Sac II (ccgcgg)位點,得到攜帶有 Sac I 位點、BspT104 I 位點、Kpn I (ggtacc)位點、SacII (ccgcgg)位點及Sac II (ccgcgg)位點的VP6編碼基因;
7)、用與步驟2)相同的PCR擴增、酶切和連接,使用對應的PETL5/P308R和P308F/PVP6-3引物對及內切酶Nde I/Xho I和Xho I/BamH I,在步驟6)所得攜帶有Sac I位點、BspT104 I 位點、Kpn I (ggtacc)位點、Bln I (ccgcgg)位點及 Sac II (ccgcgg)位點的VP6編碼基因上插入Xho I (ctcgag)位點,得到攜帶有Sac I位點、BspT104 I位點、KpnI (ggtacc)位點、Bln I (ccgcgg)位點、Sac II (ccgcgg)位點及 Xho I (ctcgag)位點的VP6載體蛋白編碼基因,該VP6F載體蛋白編碼基因由1146個核苷酸組成,前端為編碼蛋氨酸(M)的起始秘密子ATG,末端由2個終止密碼子(TGA TAA)組成,其核苷酸序列為
Iatggatgttt tgtattcatt atcaaaaact cttaaggatg ctagagacaa 51 aattgtcgaa ggcactttat actctaatgt tagtgatcta attcaacaat 101 ttaatcaaat gataattact atgaatggaa atgaatttca aactggagga 151 attggtaatc taccaaccag aaattggagt tttgattttg gtttacttgg 201 aactacgctt ttgaatttag atgccaatta tgttgaaact gcgcgtaaca 251 caattgatta ttttgttgat tttgtagata acgtatgtat ggatgagatg 301 gttagagaat cacaaagaaa cggaattgca ccgcagtcag aatcacttag 351 aaaactatca gggattaagt ttaaaagaat aaattttgac aattcgtcgg 401 aatacataga aaactggaac ttgcaaaata gaagacaaag aactggtttt 451 acatttcata aaccaaacat ctttccatat tcagcttcat ttacgttaaa 501 tagatcacaa cctgcgcatg_agctcatggg tacaatgtgg ttgaatgcag 551 gttcagagat tcaagttgct ggatttgatt attcRaacat acagcaattt 601 gagcatatag ttcagctccg tagggtgtta acgacggcta caataactct 651 tttaccagat gctgaaaggt ttagtttccc tagagtaatt aattcagctg 701 atRRtaccac atggtatttt aatccagtaa tacttagacc gaataatgtc 751 gaagtagaat ttctattgaa tggacagata ataaatactt atcaggcacc 801 taggtttggt acaattgtag ctagaaattt tgatactatt agattatcat 851 ttcaattgat gagaccgcgg accccatcag tcRcaRcact CRaRcatcat 901 gctactgtag gactaacatt acgcattgaa tctgcaattt gtgagtcagt 951 gcttgctgat gctagtgaaa caatgttagc taatgtaaca tctgttagac1001 aagaatatgc aataccagtt gggccagttt ttccaccagg tatgaattgg1051 actgatttga ttactaacta ctcaccatct agagaggata atttacaacg1101 tgtgtttaca gtagcttcca ttagaagcat gcttgttaaa tgataa
8)、將步驟7)得到的VP6載體蛋白編碼基因用常規(guī)的基因克隆和重組技術,連接到ρΕΤ表達質粒DNA的Nde I/BamH I位點,得到攜帶VP6F載體蛋白編碼基因的重組質粒pET6F。
所述步驟2 )的輪狀病毒TB-Chen株為公知的病毒株。本發(fā)明具有下列優(yōu)點和效果采用上述方案獲得的各個攜帶乙肝病毒不同抗原表位的重組蛋白,經免疫學Western B lot檢測,結果表明,所述各個重組蛋白不僅具有VP6F蛋白的抗原反應性與免疫原性,還具有HBV抗原表位的抗原反應性和免疫原性,且不具有傳染性,沒有毒力返祖和腸套疊等副作用,對開發(fā)新一代RV/HBV重組嵌合疫苗及試劑具有重要價值。
具體實施例方式實施例I
通過下列步驟構建攜帶有Sac I位點、BspT104 I位點、Kpn I位點、Bln I位點、SacII位點及Xho I位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒PETP6F 1)人工設計下列引物對
P174F CATGAGCTCATGGGTACAATGTGG ;
P174R CATGAGCTCATGCGCAGGTTGTGA ;
P197F GATTATTCGAACATACAGCAATTTGAGCAT ;
P197R TATGTTCGAATAATCAAATCCAGCAAC ;
P244F GATGGTACCACATGGTATTTTAATCCA ;
P244R ; TGTGGTACCATCAGCTGAATTAATTAC ;
P275F GCACCTAGGTTTGGTACAATTGTA ;
P275R AAACCTAGGTGCCTGATAAGTATTTAT ;
P298F AGACCGCGGACCCCATCAGTCGCA ;
P298R GGTCCGCGGTCTCATCAATTGAAATGA ;
P308F GCACTCGAGCATCATGCTACTGTA ;
P308R ATGCTCGAGTGCTGCGACTGATGG ;
以及下列引物
PETL5 ATCATATGGAGGTTCTGTACTC ;
PVP6-3 TAGGATCCTTATCATTTAACAAGCATGCTTCTAAT ;
2)以公知的輪狀病毒TB-Chen株VP6蛋白編碼基因為模板,以下列為引物對,進行PCR擴增PETL5/P174R 和 P174F/PVP6-3 ;
PETL5/P197R 和 P197F/PVP6-3 ;
PETL5/P244R 和 P244F/PVP6-3 ;
PETL5/P275R 和 P275F/PVP6-3 ;
PETL5/P298R 和 P298F/PVP6-3 ;PETL5/P308R 和 P308F/PVP6-3 ;
具體是
在PCR反應體系為PCR反應混合液體積為100 μ I,包含30ng模板DNA,I X IO5 pmol的dNTPs, IOX PCR緩沖液 10μ 1,5U Taq DNA聚合酶,60pmol 的引物對PETL5/P174R和P174F/PVP6-3,其余用水補足;PCR反應條件為94°C預變性3 min,94°C變性40s,56°C復性30s,72°C延伸反應lmin,共40個循環(huán),最后72°C延伸10 min ;得到帶有NdeI和Sac I位點序列的PCR擴增片段6174F和帶有Sac I和BamH I位點序列的PCR擴增片段6174R ;用I. 5%的瓊脂糖凝膠電泳分離和純化該擴增片段;將PCR擴增片段6174F和6174R分別用Nde I/Sac I 和 Sac I/BamH I 雙酶切,即
A、6174F酶切片段制備酶切反應體系為3μI的6174F片段,3 μ I的Nde I (15U),3μ I的Sac I (15U),8y I的IOX緩沖液,63 μ I的水;將酶切混合液分別于37°C水浴反 應3小時后,得酶切片段,用I. 5%的瓊脂糖凝膠電泳分離和純化該酶切片段;
B、除使用SacI/BamH I雙酶切外,6174R酶切片段制備與6174F酶切片段相同;
C、酶切片段的連接將6174F和6174R酶切片段與T4DNA連接酶及緩沖液混合后,在18°C連接反應6h,得到攜帶有Sac I位點(gagctc)的VP6編碼基因
3)運用步驟2)的PCR擴增、酶切和連接,使用對應的PETL5/P197R和P197F/PVP6-3引物對及內切酶Nde I/BspT104 I和BspT104 I/BamH I,在步驟2)獲得的攜帶有Sac I位點的VP6編碼基因上插入BspT104 I (ttcgaa)位點,得到攜帶有Sac I位點和BspT104 I位點的VP6編碼基因;
4 )用與步驟2 )相同的PCR擴增、酶切和連接,使用對應的PETL5/P244R和P244F/PVP6-3引物對及內切酶Nde I/Kpn I和Kpn I/BamH I,在步驟3)所得攜帶有Sac I位點和BspT104 I位點的VP6編碼基因上插入Kpn I (ggtacc)位點,得到攜帶有Sac I位點、BspT104 I位點及Kpn I (ggtacc)位點的VP6編碼基因;
5)用與步驟2)相同的PCR擴增、酶切和連接,使用對應的PETL5/P275R和P275F/PVP6-3引物對及內切酶Nde I/Bln I和Bin I/BamH I,在步驟4)所得攜帶有Sac I位點、BspT104 I位點及Kpn I (ggtacc)位點的VP6編碼基因上插入Bln I (cctagg)位點,得到攜帶有 Sac I 位點、BspT104 I 位點、Kpn I (ggtacc)位點及 Bln I (cctagg)位點的 VP6編碼基因;
6 )用與步驟2 )相同的PCR擴增、酶切和連接,使用對應的PETL5/P298R和P298F/PVP6-3引物對及內切酶Nde I/Sac II和Sac I I/BamH I,在步驟5)所得攜帶有Sac I位點、BspT104 I位點、Kpn I (ggtacc)位點及Bln I (cctagg)位點的VP6編碼基因上插入Sac II (ccgcgg)位點,得到攜帶有 Sac I 位點、BspT104 I 位點、Kpn I (ggtacc)位點、Sac
II(ccgcgg)位點及Sac II (ccgcgg)位點的VP6編碼基因;
7)、用與步驟2)相同的PCR擴增、酶切和連接,使用對應的PETL5/P308R和P308F/PVP6-3引物對及內切酶Nde I/Xho I和Xho I/BamH I,在步驟6)所得攜帶有Sac I位點、BspT104 I 位點、Kpn I (ggtacc)位點、Bln I (ccgcgg)位點及 Sac II (ccgcgg)位點的VP6編碼基因上插入Xho I (ctcgag)位點,得到攜帶有Sac I位點、BspT104 I位點、Kpn
I(ggtacc)位點、Bln I (ccgcgg)位點、Sac II (ccgcgg)位點及 Xho I (ctcgag)位點的VP6載體蛋白編碼基因,該VP6F載體蛋白編碼基因由1146個核苷酸組成,前端為編碼蛋氨酸(M)的起始秘密子ATG,末端由2個終止密碼子(TGA TAA)組成,其核苷酸序列為
I atggatgttt tgtattcatt atcaaaaact cttaaggatg ctagagacaa51 aattgtcgaa ggcactttat actctaatgt tagtgatcta attcaacaat101 ttaatcaaat gataattact atgaatggaa atgaatttca aactggagga151 attggtaatc taccaaccag aaattggagt tttgattttg gtttacttgg201 aactacgctt ttgaatttag atgccaatta tgttgaaact gcgcgtaaca251 caattgatta ttttgttgat tttgtagata acgtatgtat ggatgagatg301 gttagagaat cacaaagaaa cggaattgca ccgcagtcag aatcacttag351 aaaactatca gggattaagt ttaaaagaat aaattttgac aattcgtcgg401 aatacataga aaactggaac ttgcaaaata gaagacaaag aactggtttt451 acatttcata aaccaaacat ctttccatat tcagcttcat ttacgttaaa501 tagatcacaa cctgcgcatg_agctcatggg tacaatgtgg ttgaatgcag551 gttcagagat tcaagttgct ggatttgatt attcgaacat acagcaattt 601 gagcatatag ttcagctccg tagggtgtta acgacggcta caataactct651 tttaccagat gctgaaaggt ttagtttccc tagagtaatt aattcagctg701 atggtaccac atggtatttt aatccagtaa tacttagacc gaataatgtc751 gaagtagaat ttctattgaa tggacagata ataaatactt atcaggcacc801 taggtttggt acaattgtag ctagaaattt tgatactatt agattatcat851 ttcaattgat gagaccgcgg accccatcag tcgcagcact cgagcatcat901 gctactgtag gactaacatt acgcattgaa tctgcaattt gtgagtcagt951 gcttgctgat gctagtgaaa caatgttagc taatgtaaca tctgttagac
1001 aagaatatgc aataccagtt gggccagttt ttccaccagg tatgaattgg
1051 actgatttga ttactaacta ctcaccatct agagaggata atttacaacg
1101 tgtgtttaca gtagcttcca ttagaagcat gcttgttaaa tgataa
8)、將步驟7)得到的VP6載體蛋白編碼基因用常規(guī)的基因克隆和重組技術,連接到ρΕΤ表達質粒DNA的Nde I/BamH I位點,得到攜帶VP6F載體蛋白編碼基因的重組質粒pET6F。實施例2
本發(fā)明提供的攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白通過下列步驟制備 A、根據(jù)抗原表位氨基酸序列,分別設計下列引物
S82F CTTCCTCTTCATCCTGCTGCTATGCCTGGAGCT
S82R CCAGGCATAGCAGCAGGATGAAGAGGAAGAGCT
B、將步驟A的引物進行如下配對將S抗原表位S82的兩條人工合成的寡核苷酸即正鏈S82F和負鏈S82R配成引物對S82F/S82R ;C、將步驟B的引物對在下列條件下進行退火,退火混合液中分別含有2μI的正向引物,2 μ I的反向引物,以及7 μ I的PCR緩沖液和59 μ I的水;將退火混合液在65°C水浴反應5分鐘后,放入37°C水浴反應45分鐘,反應體系中加入400 μ I的TE,并與等體積異丙醇充分混勻,室溫放置10分鐘后,4°C,15,OOOrpm離心15min,沉淀用質量濃度為75%的乙醇漂洗,干燥后,用IOyl的TE溶解;得到在5’端和3’端分別帶Sac I位點的互補雙鏈S82互補雙鏈;
D、將攜帶有SacI位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒pETP6F,在下列條件下用Sac I酶切酶切混合液含3 μ I的Sac I內切酶(15U),以及10 μ I的pETP6FDNA, 7 μ I的10 X緩沖液,50 μ I的水;將酶切混合液放于37°C水浴反應3小時,得Sac I酶切質粒片段;用I. 5%的瓊脂糖凝膠電泳分離和純化所得酶切質粒片段;
E、將步驟D所得酶切質粒片段與步驟C所得互補雙鏈配對如下 Sac I酶切質粒片段/S82互補雙鏈; 將上述配對的片段與T4 DNA連接酶及緩沖液混合后,在18°C連接反應6h,得到在SacI位點攜帶有S82抗原表位基因的重組質粒pETP6F/S82 ;按常規(guī)分別將所得重組質粒經菌體轉化擴增后,進行酶切鑒定和核苷酸序列測定;
F、將步驟E得到的重組質粒,即PETP6F/S82,在下列條件下分別轉化BL21(DE3)感受態(tài)細胞10μ I的重組質粒與100 μ I BL21 (DE3)感受態(tài)細胞混勻,冰上放置30分鐘,42°C放置70秒鐘,加入500 μ I LB培養(yǎng)液后,在37°C培養(yǎng)30分鐘,培養(yǎng)板涂含200 μ g/ml氨芐青霉素的I. 5%瓊脂培養(yǎng)板,培養(yǎng)板于37°C培養(yǎng)14個小時,挑取單個菌落放入含200 μ g/ml氨芐青霉素的LB培養(yǎng)液中培養(yǎng)和表達,得到攜帶乙肝病毒S82抗原表位的重組蛋白rVP6/S82。實施例3
A、引物設計為
C19F CGAATCATTTTTGCCTTCTGACTTCTTTCCTTCCGTCCGGGATTTC19R CGAAATCCCGGACGGAAGGAAAGAAGTCAGAAGGCAAAAATGATT
B、引物配對為
將C抗原表位C19,其氛基酸序列為Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser ValArg Asp的兩條人工合成的寡核苷酸即正鏈(C19F)和負鏈(C19R)配成引物對C19F/C19R ;
C、同實施例2 D、將攜帶有BspT104I位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒pETP6F,在與實施例2步驟D相同條件下用BspT104 I酶切,得酶切質粒片段BspT104 I酶切質粒片段;
E、將步驟D所得酶切質粒片段與步驟C所得互補雙鏈配對如下
BspT104 I酶切質粒片段/C19互補雙鏈;
將上述配對的片段與T4 DNA連接酶及緩沖液混合后,在18°C連接反應6h,得到在BspT104 I位點攜帶有C19抗原表位基因的重組質粒pETP6F/C19 ;
F、將步驟E得到的重組質粒pETP6F/C19在與實施例2步驟F相同的條件下轉化BL21(DE3)感受態(tài)細胞,得到攜帶乙肝病毒C19抗原表位的重組蛋白rVP6F/C19。實施例4 除引物為
S125F CACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGTACS125R CATCCGTAGGTTTTGTACAGCAACATGAGGGAAACATAGAGTTGCCTTGAGCAGGAGTGGTAC酶切時將攜帶有Kpn I (ggtacc)位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒PETP6F,用Kpn I酶切外,其余均同實施例2,得到攜帶乙肝病毒S125抗原表位的重組蛋白rVP6F/S1250實施例5
除引物為
S2[8]F CTAGGTTCCACCAAGCTCTGCAAGATCCCAGAGTCAGGGGCCTGTATTTTCCTGCTGGTC
S2[8] R CTAGGACCAGCAGGAAAATACAGGCCCCTGACTCTGGGATCTTGCAGAGCTTGGTGGAAC酶切時將將攜帶有Bin I (ccgcgg)位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒pETP6F,用Bln I酶切外,其余均同實施例2,得到攜帶乙肝病毒S2[8]抗原表位的重組蛋白 rVP6F/S2[8]。實施例6 除引物為
SI[93]F GGCCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCGCSI[93] R GGCTGCCTTCCTGACTGCCGATTGGTGGAGGCAGGAGGAGGCCGC酶切時將將攜帶有Sac II (ccgcgg)位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒PETP6F,用Sac II酶切外,其余均同實施例2,得到攜帶乙肝病毒SI [93]抗原表位的重組蛋白 rVP6F/Sl[93]。實施例7 除引物為
C117F TCGAGTATTTGGTCTCTTTCGGAGTGTGGATTCGCACTCCACCAGCTTATAGACCACCAAATGCCCCTATCC
C117R TCGAGGATAGGGGCATTTGGTGGTCTATAAGCTGGTGGAGTGCGAATCCACACTCCGAAAGAGACCAAATAC ;
酶切時將將攜帶有Xho I (ctcgag)位點的重組輪狀病毒VP6載體蛋白編碼基因的質粒PETP6F,用Xho I酶切外,其余均同實施例2,得到攜帶乙肝病毒C117抗原表位的重組蛋白 rVP6F/C1170實施例8
本發(fā)明提供的同時攜帶乙肝病毒S82,C19,S125,S2[8],S1[93]和C117抗原表位的重組蛋白通過下列步驟制備A、同實施例2步驟A;
B、同實施例2步驟B;
C、同實施例2步驟C;
D、同實施例2步驟D;
E、同實施例2步驟E;
F、將步驟E所得重組質粒pETP6F/S82用BspT104I酶切,并與步驟C所得C19互補雙鏈連接,得到在Sac I和BspT104 I位點攜帶有S82和C19抗原表位基因的重組質粒pETP6F/S82/C19 ;
G、將步驟E所得重組質粒pETP6F/S82/C19用KpnI酶切,并與步驟C所得S125互補雙鏈連接,得到在Sac I,BspT104 I和Kpn I位點攜帶有S82,C19和S125抗原表位基因的重組質粒 PETP6F/S82/C19/S125 ;
H、將步驟E所得重組質粒pETP6F/S82/C19/S125用BlnI酶切,并與步驟C所得S2 [8]互補雙鏈連接,得到在Sac I,BspT104 I, Kpn I和Bin I位點攜帶有S82,C19,S125和S2[8]抗原表位基因的重組質粒pETP6F/S82/C19/S125/S2[8];
I、將步驟E所得重組質粒pETP6F/S82/C19/S125/S2[8]用Sac II酶切,并與步驟C所得Sl[93]互補雙鏈連接,得到在Sac I,BspT104 I,Kpn I,Bln I和Sac II位點攜帶有S82,C19,S125,S2[8]和 SI [93]抗原表位基因的重組質粒pETP6F/S82/C19/S125/S2 [8] /SI[93];
J、將步驟E所得重組質粒pETP6F/S82/C19/S125/S2[8]/Sl[93]用Xho I酶切,并與步驟C所得Cl 17互補雙鏈連接,得到在Sac I,BspT104 I,Kpn I,Bln I, Sac II和Xho I位點攜帶有對應的S82,C19,S125,S2[8],SI [93]和C117抗原表位基因的重組質粒pETP6F/VP⑥H ;按常規(guī)將所得重組質粒經菌體轉化擴增后,進行酶切鑒定和核苷酸序列測定;
K、將步驟J得到的重組質粒pETP6F/VP⑥H在下列條件下轉化BL21 (DE3)感受態(tài)細胞10 μ I重組質粒與100 μ I BL21 (DE3)感受態(tài)細胞混勻,并上放置30分鐘,轉42°C放置70秒鐘,加入500 μ I LB培養(yǎng)液后,在37°C培養(yǎng)30分鐘。培養(yǎng)物涂含200 μ g/ml氨芐青霉素的I. 5%瓊脂培養(yǎng)板;培養(yǎng)板放37°C培養(yǎng)箱中培養(yǎng)14個小時,挑取單個菌落放入含200 μ g/ml氨芐青霉素的LB培養(yǎng)液中培養(yǎng)和表達,得同時攜帶乙肝病毒S82,C19,S125,S2[8],S1[93]和Cl 17抗原表位的重組蛋白rVP6F⑥H。本發(fā)明所得各個攜帶乙型肝炎病毒不同抗原表位的重組嵌合蛋白,經下列免疫學Western Blot 檢測
1)將攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白rVP6/S82,攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白rVP6F/C19,攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白rVP6F/S125,攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白rVP6F/S2[8],攜帶有乙肝病毒SI [93]抗原表位的重組嵌合蛋白rVP6F/Sl[93],攜帶有乙肝病毒C117抗原表位的重組嵌合蛋白rVP6F/C117,和同時攜帶乙肝病毒S82, C19, S125,S2[8],S1[93]和C117抗原表位的重組蛋白rVP6F⑥H,以及用作對照的重組輪狀病毒NSP2蛋白,和用作對照的牛血清白蛋白,各取2μ g分別溶于20μ I水,分別加入5μ I 5Χ變性緩沖液,混勻后,在100°C變性5min ;
2)將上述I)的變性蛋白樣品分別用10%聚丙烯酰胺凝膠電泳(10%SDS-PAGE)分離,并在電壓15V、電流50mA的條件下電泳轉印25 min,轉移到PVDF膜上;將膜置于含5%脫脂奶粉的PBS/T液中于4° C封閉過夜;
3)用PBS/T洗膜5次,分別加入抗輪狀病毒全病毒豚鼠血清抗體(5%脫脂奶粉1:400稀釋),抗重組輪狀病毒VP6蛋白豚鼠血清抗體(1%脫脂奶粉1:400稀釋),抗重組輪狀病毒NSP2蛋白豚鼠血清抗體(1%脫脂奶粉1:400稀釋),HBsAb陽性的乙型肝炎患者的血清抗體(1%脫脂奶粉1:200稀釋),HBcAb陽性的乙型肝炎患者的血清抗體(1%脫脂奶粉1:200稀釋),于37°C孵育I. 5 h ;用PBS/T液洗膜5次,每次5 min ;
4)加入HRP標記的羊抗豚鼠IgG(1:2,000如上稀釋)/ HRP標記的兔抗人IgG (1:800如上稀釋),37°C孵育I h,用PBS/T漂洗5次,DAB顯色;顯色后,蒸餾水漂洗膜,終止顯色,觀察并分析結果如下 將攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白rVP6/S82,攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白rVP6F/C19,攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白rVP6F/S125,攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白rVP6F/S2[8],攜帶有乙肝病毒SI [93]抗原表位的重組嵌合蛋白rVP6F/Sl[93],攜帶有乙肝病毒C117抗原表位的重組嵌合蛋白rVP6F/Cl 17,均能與抗輪狀病毒全病毒抗體發(fā)生特異性免疫反應,能與抗輪狀病毒VP6蛋白抗體發(fā)生特異性免疫反應,不能與抗重組輪狀病毒NSP2蛋白豚鼠血清抗體發(fā)生特異性免疫反應。
將攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白rVP6/S82,攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白rVP6F/S125,攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白rVP6F/S2 [8],攜帶有乙肝病毒SI [93]抗原表位的重組嵌合蛋白rVP6F/Sl [93],能與HBsAb陽性的乙型肝炎患者的血清抗體發(fā)生特異性免疫反應。攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白rVP6F/C19,攜帶有乙肝病毒C117抗原表位的重組嵌合蛋白rVP6F/C117,能與HBcAb陽性的乙型肝炎患者的血清抗體發(fā)生特異性免疫反應。同時攜帶乙肝病毒S82,C19,S125,S2[8],S1[93]和C117抗原表位的重組蛋白rVP6F⑥H,能與抗輪狀病毒全病毒抗體發(fā)生特異性免疫反應,能與抗輪狀病毒VP6蛋白抗體發(fā)生特異性免疫反應,能與HBsAb陽性的乙型肝炎患者的血清抗體發(fā)生特異性免疫反應,能與HBcAb陽性的乙型肝炎患者的血清抗體發(fā)生特異性免疫反應,不能與抗重組輪狀病毒NSP2蛋白豚鼠血清抗體發(fā)生特異性免疫反應。用作陰性對照的重組輪狀病毒NSP2蛋白只能與抗重組輪狀病毒NSP2蛋白豚鼠血清抗體發(fā)生特異性免疫反應。用作陰性對照的牛血清白蛋白,不能與上述所有檢測抗體發(fā)生特異性免疫反應。結論上述所有的重組嵌合蛋白均具有載體蛋白和所攜帶的抗原表位特異的抗原反應性。序列表
攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白的氨基酸序列
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Phe Leu Phe IleLeu Leu Leu Cys Leu Glu Leu Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu He GlnVal Ala Gly Phe Asp Tyr Ser Asn He Gln Gln Phe Glu His He Val Gln Leu Arg ArgVal Leu Thr Thr Ala Thr He Thr Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro ArgVal He Asn Ser Ala Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val He Leu Arg Pro AsnAsn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile He Asn Thr Tyr Gln Ala Pro ArgPhe Gly Thr He Val Ala Arg Asn Phe Asp Thr He Arg Leu Ser Phe Gln Leu Met ArgPro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val Gly Leu Thr Leu ArgHe Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala AsnVal Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly MetAsn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg ValPhe Thr Val Ala Ser lie Arg Ser Met Leu Val Lys
抗原表位 S82 的氨基酸序列Phe Leu Phe lie Leu Leu Leu Cys Leu 攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白的氨基酸序列 Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn Ser Phe LeuPro Ser Asp Phe Phe Pro Ser Val Arg Asp Ser Asn He Gln Gln Phe Glu His He ValGln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu Pro Asp Ala Glu Arg PheSer Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val IleLeu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln He He Asn Thr TyrGln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp Thr He Arg Leu Ser PheGln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val GlyLeu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu ThrMet Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val PhePro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp AsnLeu Gln Arg Val Phe Thr Val Ala Ser He Arg Ser Met Leu Val Lys抗原表位C19的氨基酸序列
Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白的氨基酸序列
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn lie Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr lie Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val lie Asn Ser Ala Asp Gly Thr Thr Pro AlaGln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Thr Thr TrpTyr Phe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn GlyGln He He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr lie Val Ala Arg Asn Phe AspThr He Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu GluHis His Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala lie Cys Glu Ser Val LeuAla Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala liePro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu lie Thr Asn Tyr SerPro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser lie Arg Ser Met LeuVal Lys
抗原表位S125的氨基酸序列
Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro ThrAsp Gly
攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白的氨基酸序列
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnIle He Asn Thr Tyr Gln Ala Pro Arg Phe His Gln Ala Leu Gln Asp Pro Arg Val ArgGly Leu Tyr Phe Pro Ala Gly Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp ThrHe Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu HisHis Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu AlaAsp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala He ProVal Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr Ser ProSer Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser He Arg Ser Met Leu ValLys
抗原表位S2[8]的氨基酸序列
Pro Arg Phe His Gln Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro AlaGly
攜帶有乙肝病毒Sl[93]抗原表位的重組嵌合蛋白的氨基酸序列
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp T yr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnIle He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp ThrHe Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Pro Pro Pro Ala Ser Thr Asn Arg GlnSer Gly Arg Gln Pro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val GlyLeu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu ThrMet Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val PhePro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp AsnLeu Gln Arg Val Phe Thr Val Ala Ser He Arg Ser Met Leu Val Lys
抗原表位SI [93]的氨基酸序列
Pro Pro Pro Ala Ser Thr Asn Arg Gln Ser Gly Arg Gln0攜帶有乙肝病毒Cl 17抗原表位的重組嵌合蛋白的氨基酸序列
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnIle He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp ThrIle Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu TyrLeu Val Ser Phe Gly Val Trp He Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala ProHe Leu Glu His His Ala Thr Val Gly Leu Thr Leu Arg lie Glu Ser Ala lie Cys GluSer Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln GluTyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu lie ThrAsn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser lie ArgSer Met Leu Val Lys
抗原表位Cl 17的氨基酸序列
Tyr Leu Val Ser Phe Gly Val Trp He Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro lie
同時攜帶乙肝病毒S82,C19,S125,S2[8],SI [93]和C117抗原表位的重組蛋白的氨基酸序列
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Phe Leu Phe IleLeu Leu Leu Cys Leu Glu Leu Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu He GlnVal Ala Gly Phe Asp Tyr Ser Asn Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val ArgAsp Ser Asn He Gln Gln Phe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr AlaThr He Thr Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser AlaAsp Gly Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys ProThr Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu ValGlu Phe Leu Leu Asn Gly Gln He He Asn Thr Tyr Gln Ala Pro Arg Phe His Gln AlaLeu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Pro Arg Phe Gly Thr IleVal Ala Arg Asn Phe Asp Thr He Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Pro ProPro Ala Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Arg Thr Pro Ser Val Ala Ala LeuGlu Tyr Leu Val Ser Phe Gly Val Trp He Arg Thr Pro Pro Ala Tyr Arg Pro Pro AsnAla Pro He Leu Glu His His Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala IleCys Glu Ser Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val ArgGln Glu Tyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp LeuHe Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala SerHe Arg Ser Met Leu Val Lys引物
S82F CTTCCTCTTCATCCTGCTGCTATGCCTGGAGCTS82R CCAGGCATAGCAGCAGGATGAAGAGGAAGAGCT
C19F CGAATCATTTTTGCCTTCTGACTTCTTTCCTTCCGTCCGGGATTTC19R CGAAATCCCGGACGGAAGGAAAGAAGTCAGAAGGCAAAAATGATTS125F CACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGTACS125R CATCCGTAGGTTTTGTACAGCAACATGAGGGAAACATAGAGTTGCCTTGAGCAGGAGTGGTACS2[8] F CTAGGTTCCACCAAGCTCTGCAAGATCCCAGAGTCAGGGGCCTGTATTTTCCTGCTGGTCS2[8] R CTAGGACCAGCAGGAAAATACAGGCCCCTGACTCTGGGATCTTGCAGAGCTTGGTGGAACSI[93]F GGCCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCGCSI[93] R GGCTGCCTTCCTGACTGCCGATTGGTGGAGGCAGGAGGAGGCCGCC117F TCGAGTATTTGGTCTCTTTCGGAGTGTGGATTCGCACTCCACCAGCTTATAGACCACCAAATGCCCCTATCC
C117R TCGAGGATAGGGGCATTTGGTGGTCTATAAGCTGGTGGAGTGCGAATCCACACTCCGAAAGAGACCA AATAC
攜帶有 Sac I 位點、BspT104 I 位點、Kpn I (ggtacc)位點、Bin I (ccgcgg)位點、Sac
II(ccrcrr)位點及Xho I (ctcRaR)位點的VP6載體蛋白編碼基因的核苷酸序列:
I atggatgttt tgtattcatt atcaaaaact cttaaggatg ctagagacaa 51 aattgtcgaa ggcactttat actctaatgt tagtgatcta attcaacaat 101 ttaatcaaat gataattact atgaatggaa atgaatttca aactggagga 151 attggtaatc taccaaccag aaattggagt tttgattttg gtttacttgg 201 aactacgctt ttgaatttag atgccaatta tgttgaaact gcgcgtaaca 251 caattgatta ttttgttgat tttgtagata acgtatgtat ggatgagatg 301 gttagagaat cacaaagaaa cggaattgca ccgcagtcag aatcacttag 351 aaaactatca gggattaagt ttaaaagaat aaattttgac aattcgtcgg 401 aatacataga aaactggaac ttgcaaaata gaagacaaag aactggtttt 451 acatttcata aaccaaacat ctttccatat tcagcttcat ttacgttaaa 501 tagatcacaa cctgcgcatg_agctcatggg tacaatgtgg ttgaatgcag 551 gttcagagat tcaagttgct ggatttgatt attcRaacat acagcaattt 601 gagcatatag ttcagctccg tagggtgtta acgacggcta caataactct 651 tttaccagat gctgaaaggt ttagtttccc tagagtaatt aattcagctg 701 atRRtaccac atggtatttt aatccagtaa tacttagacc gaataatgtc 751 gaagtagaat ttctattgaa tggacagata ataaatactt atcaggcacc 801 taggtttggt acaattgtag ctagaaattt tgatactatt agattatcat 851 ttcaattgat gagaccgcgg accccatcag tcRcaRcact CRaRcatcat 901 gctactgtag gactaacatt acgcattgaa tctgcaattt gtgagtcagt 951 gcttgctgat gctagtgaaa caatgttagc taatgtaaca tctgttagac 1001 aagaatatgc aataccagtt gggccagttt ttccaccagg tatgaattgg 1051 actgatttga ttactaacta ctcaccatct agagaggata atttacaacg 1101 tgtgtttaca gtagcttcca ttagaagcat gcttgttaaa tgataa。
權利要求
1.一種攜帶有乙肝病毒S82抗原表位的重組嵌合蛋白,其特征在于其氨基酸序列為Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Phe Leu Phe IleLeu Leu Leu Cys Leu Glu Leu Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu He GlnVal Ala Gly Phe Asp Tyr Ser Asn He Gln Gln Phe Glu His He Val Gln Leu Arg ArgVal Leu Thr Thr Ala Thr He Thr Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro ArgVal He Asn Ser Ala Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val He Leu Arg Pro AsnAsn Val Glu Val Glu Phe Leu Leu Asn Gly Gln He He Asn Thr Tyr Gln Ala Pro ArgPhe Gly Thr He Val Ala Arg Asn Phe Asp Thr He Arg Leu Ser Phe Gln Leu Met ArgPro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val Gly Leu Thr Leu ArgHe Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala AsnVal Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly MetAsn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg ValPhe Thr Val Ala Ser He Arg Ser Met Leu Val Lys ;其中,所述抗原表位S82的氨基酸序列為Phe Leu Phe He Leu Leu Leu Cys Leu。
2.—種攜帶有乙肝病毒C19抗原表位的重組嵌合蛋白,其特征在于其氨基酸序列為Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn Ser Phe LeuPro Ser Asp Phe Phe Pro Ser Val Arg Asp Ser Asn He Gln Gln Phe Glu His He ValGln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu Pro Asp Ala Glu Arg PheSer Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp Tyr Phe Asn Pro Val IleLeu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln I Ie He Asn Thr TyrGln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp Thr He Arg Leu Ser PheGln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val GlyLeu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu ThrMet Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala lie Pro Val Gly Pro Val PhePro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp AsnLeu Gln Arg Val Phe Thr Val Ala Ser lie Arg Ser Met Leu Val Lys ; 其中,所述抗原表位C19的氨基酸序列為Ser Phe Leu Pro Ser Asp Phe Phe Pro SerVal Arg Asp。
3.一種攜帶有乙肝病毒S125抗原表位的重組嵌合蛋白,其特征在于其氨基酸序列為Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Pro AlaGln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Thr Thr TrpTyr Phe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn GlyGln He He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe AspThr He Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu GluHis His Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val LeuAla Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala IlePro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr SerPro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser He Arg Ser Met LeuVal Lys ; 其中,所述抗原表位S125的氨基酸序列為Thr Thr Pro Ala Gln Gly Asn Ser MetPhe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly0
4.一種攜帶有乙肝病毒S2[8]抗原表位的重組嵌合蛋白,其特征在于其氨基酸序列為
5.—種攜帶有乙肝病毒SI [93]抗原表位的重組嵌合蛋白,其特征在于其氨基酸序列為Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnHe He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp ThrHe Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Pro Pro Pro Ala Ser Thr Asn Arg GlnSer Gly Arg Gln Pro Arg Thr Pro Ser Val Ala Ala Leu Glu His His Ala Thr Val GlyLeu Thr Leu Arg He Glu Ser Ala He Cys Glu Ser Val Leu Ala Asp Ala Ser Glu ThrMet Leu Ala Asn Val Thr Ser Val Arg Gln Glu Tyr Ala He Pro Val Gly Pro Val PhePro Pro Gly Met Asn Trp Thr Asp Leu He Thr Asn Tyr Ser Pro Ser Arg Glu Asp AsnLeu Gln Arg Val Phe Thr Val Ala Ser He Arg Ser Met Leu Val Lys ;其中,所述抗原表位SI [93]的氨基酸序列為Pro Pro Pro Ala Ser Thr Asn Arg GlnSer Gly Arg Gin。
6.一種攜帶有乙肝病毒C117抗原表位的重組嵌合蛋白,其特征在于其氨基酸序列為Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Met Gly Thr MetTrp Leu Asn Ala Gly Ser Glu He Gln Val Ala Gly Phe Asp Tyr Ser Asn He Gln GlnPhe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr He Thr Leu Leu ProAsp Ala Glu Arg Phe Ser Phe Pro Arg Val He Asn Ser Ala Asp Gly Thr Thr Trp TyrPhe Asn Pro Val He Leu Arg Pro Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly GlnIle He Asn Thr Tyr Gln Ala Pro Arg Phe Gly Thr He Val Ala Arg Asn Phe Asp ThrHe Arg Leu Ser Phe Gln Leu Met Arg Pro Arg Thr Pro Ser Val Ala Ala Leu Glu TyrLeu Val Ser Phe Gly Val Trp He Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala ProHe Leu Glu His His Ala Thr Val Gly Leu Thr Leu Arg He Glu Ser Ala He Cys GluSer Val Leu Ala Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln GluTyr Ala He Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr Asp Leu He ThrAsn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg Val Phe Thr Val Ala Ser He ArgSer Met Leu Val Lys ; 其中,所述抗原表位C117的氨基酸序列為Tyr Leu Val Ser Phe Gly Val Trp IleArg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro lie。
7.—種同時攜帶乙肝病毒S82,C19,S125,S2[8],S1[93]和C117抗原表位的重組嵌合蛋白,其特征在于其氨基酸序列為Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys He ValGlu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu He Gln Gln Phe Asn Gln Met He IleThr Met Asn Gly Asn Glu Phe Gln Thr Gly Gly He Gly Asn Leu Pro Thr Arg Asn TrpSer Phe Asp Phe Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val GluThr Ala Arg Asn Thr He Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys Met Asp GluMet Val Arg Glu Ser Gln Arg Asn Gly He Ala Pro Gln Ser Glu Ser Leu Arg Lys LeuSer Gly He Lys Phe Lys Arg He Asn Phe Asp Asn Ser Ser Glu Tyr He Glu Asn TrpAsn Leu Gln Asn Arg Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn He Phe ProTyr Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Glu Leu Phe Leu Phe IleLeu Leu Leu Cys Leu Glu Leu Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu He GlnVal Ala Gly Phe Asp Tyr Ser Asn Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val ArgAsp Ser Asn He Gln Gln Phe Glu His He Val Gln Leu Arg Arg Val Leu Thr Thr AlayTB八 ΠΘ1J8§ Sjy ΘΤΙ-ieS T3IV PA 8Md VeK s^V naq usy dsy njg Sjy jag ojj jagusy Jiyi ajineq dsy Jiu djX usV 識W ^IO ojj ojj 8 ojj ojj θπ 可v no uIOS- γ PA J9s Jiu T13A usy T3JV naq 識鞏 nIO jf5S ^IV dsy ejy naqJ9S no sjiD^II 可V no ^II Sjy naq naq A13 χτ3Λ 13IV sTH siH ηχο naq θπ ojd ejyusy ojj ojj Sjy αΑχ \γ ojj ojj Jtyi Sjy Θχι dj;x χ 3Λ Ajg 8 jagnaq αΑχ nj0naq ejy ejy p八 jag ojj Jtyi Sjy ojj iqg Sjy Ajg jag ujg Sjy usy Jiyijag ejy ojjojj ojj Sjy ojj Sjy neq ujg eiy neq Sjy 8jj Ji|x dsy 8 usySjy Bjy ■[它ΛθΙΙ *1^1 ^Md sjV 0^d ^IO ^IV OJj eiy neq Sjy ρΛ 0 dsy iqo neqT3IV UJO SIH ^Md Sjy ojj ejy ujo αΑχ usy Θχι θπ ujo Ajg usy naq naq 8 ηχο PA 可3 ρλ usy usy ojj Sjy naq aji 嚇八 0 usy 8 αΑχ djxJi\o dsy0 s句 jqx sAj sAj sAj J0S 0 8 %d]f( Jas usy ujo ejy 0 Ji\o dsy
全文摘要
本發(fā)明提供一種攜帶乙肝病毒抗原表位的重組嵌合蛋白及其制備,具體是分別攜帶有乙肝病毒S82、C19、S125、S2[8]、S1[93]或C117抗原表位的重組嵌合蛋白,以及同時攜帶乙肝病毒S82,C19,S125,S2[8],S1[93]和C117抗原表位的重組嵌合蛋白,經免疫學WesternBlot檢測,所述各個重組蛋白不僅具有VP6F蛋白的抗原反應性與免疫原性,還具有HBV抗原表位的抗原反應性和免疫原性,且不具有傳染性,沒有毒力返祖和腸套疊等副作用,對開發(fā)新一代RV/HBV重組嵌合疫苗及試劑具有重要價值。
文檔編號C12N15/70GK102643349SQ20121011259
公開日2012年8月22日 申請日期2012年4月17日 優(yōu)先權日2012年4月17日
發(fā)明者陳元鼎 申請人:中國醫(yī)學科學院醫(yī)學生物學研究所
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