CN114957411A - 一种正戊型肝病毒属基因c1型病毒orf2重组蛋白及其制备方法和应用 - Google Patents
一种正戊型肝病毒属基因c1型病毒orf2重组蛋白及其制备方法和应用 Download PDFInfo
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- CN114957411A CN114957411A CN202210574792.8A CN202210574792A CN114957411A CN 114957411 A CN114957411 A CN 114957411A CN 202210574792 A CN202210574792 A CN 202210574792A CN 114957411 A CN114957411 A CN 114957411A
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Abstract
本发明提供了一种正戊型肝病毒属基因C1型病毒ORF2重组蛋白及其制备方法和应用，属于戊型肝炎病毒技术领域，本发明提供的大鼠HEV‑C1ORF2重组蛋白，氨基酸序列如SEQIDNo.1所示；所述大鼠HEV‑C1ORF2重组蛋白能够最大限度地保留大鼠HEV的免疫原性；所述重组蛋白能够刺激不同物种动物产生特异性抗体。免疫HEV‑C1ORF2蛋白的大鼠血清中产生的针对HEV‑C1ORF2的特异性抗体能有效阻断HEV‑C1ORF2病毒样颗粒与大鼠肝癌细胞RH‑35的特异性结合，证实所述重组蛋白能够用于制备HEV‑C1疫苗。
Description
技术领域
本发明属于戊型肝炎病毒技术领域，尤其涉及一种正戊型肝病毒属基因C1 型病毒ORF2重组蛋白及其制备方法和应用。
背景技术
戊型肝炎病毒(Hepatitis E virus，HEV)是一种无包膜的正单链RNA病毒，已被分类为戊型肝炎病毒科(Hepeviridae)家族正肝病毒属(Orthohepevirus) (Smith DB,Simmonds P,Members Of The International Committee On The Taxonomy OfVirusesStudy G,Jameel S,Emerson SU,Harrison TJ,et al.Consensus proposals forclassification of the family Hepeviridae.J Gen Virol.2014Oct；95(Pt 10):2223-32.)。正肝病毒属包括HEV-A至HEV-D四个型(SmithDB,Simmonds P,Izopet J,Oliveira-Filho EF,Ulrich RG,Johne R,et al.Proposed reference sequences for hepatitis Evirus subtypes.J Gen Virol.2016Mar；97(3):537-42.)，其中HEV-A主要感染人类，引起人类的戊型肝炎感染，而HEV-C1主要感染大鼠 (Reuter G,Boros A,Pankovics P.Reviewof Hepatitis E Virus in Rats:Evident Risk of Species Orthohepevirus C toHuman Zoonotic Infection and Disease.Viruses.2020 Oct 9；12(10).)，称之为大鼠HEV(rat HEV,rHEV)。2010年，大鼠HEV首次在德国的野生大鼠中发现(Johne R,Heckel G,Plenge-Bonig A,Kindler E,Maresch C,Reetz J,et al.Novel hepatitis E virusgenotype in Norway rats,Germany.Emerg InfectDis.2010Sep；16(9):1452-5.)，随后在欧洲、亚洲和美国分离发现(Johne R, Dremsek P,Reetz J,Heckel G,Hess M,UlrichRG.Hepeviridae:an expanding family ofvertebrate viruses.Infect GenetEvol.2014Oct；27:212-29.)。以前的研究表明， rHEV发生跨宿主感染的可能性极低。但是，2018年在一个肝脏移植病人的体内发现了大鼠HEV病毒基因组。随后，在免疫缺陷型的病人患者中发现大鼠HEV 能够引起急性肝炎，并在多个地区发现了人感染HEV的病例 (SridharS,Yip CC,Wu S,ChewNF,Leung KH,Chan JF,et al.Transmission of Rat Hepatitis EVirus Infection to Humans in Hong Kong:A Clinical and EpidemiologicalAnalysis.Hepatology.2021Jan；73(1):10-22.；Sridhar S,Yip CCY,Wu S,Cai J,ZhangAJ,Leung KH,et al.Rat Hepatitis E Virus as Cause of Persistent Hepatitisafter Liver Transplant.Emerg Infect Dis.2018Dec；24(12):2241-50.)，说明大鼠HEV能够感染人类，具有人畜共患的特征。
大鼠HEV与其他HEV病毒一样，含有三个开放阅读框(Opening Reading Frame，ORF)，分别编码ORF1、ORF2和ORF3。ORF1为非结构蛋白，参与病毒复制。ORF2编码病毒衣壳蛋白ORF2，主要参与病毒颗粒的装配、与宿主细胞结合并诱导宿主产生中和抗体。ORF3编码多功能磷酸蛋白ORF3，主要参与病毒粒子的释放。大鼠HEV能编码ORF4，与ORF1的C端相重叠，其功能未知。
ORF2具有三种不同的形式，分别是感染型ORF2、糖基化ORF2和截短的 ORF2，其中感染型ORF2主要存在病毒颗粒中，而糖基化和截短的ORF2为ORF2 的主要分泌形式(Yin X,Ying D,Lhomme S,Tang Z,Walker CM,Xia N,et al. Origin,antigenicity,andfunction of a secreted form of ORF2 in hepatitis E virus infection.Proc NatlAcad Sci U SA.2018May 1；115(18):4773-8.)。ORF2衣壳蛋白高度保守，免疫原性强。ORF2能够刺激机体产生体液免疫应答，也是细胞免疫应答的主要靶标。在病人的血清中能够检测到ORF2刺激产生的相应抗体。
由于HEV-A与HEV-C1亲缘关系较远，其ORF2的同源性仅为50％～60％，提示人ORF2与大鼠ORF2的免疫原性相差较大，因而人HEV的ORF2蛋白与大鼠 HEV感染人体之后产生的针对ORF2的抗体之间的交叉反应性较小，不能用于检测感染大鼠HEV的病人的血清样本，也不能应用于研发大鼠HEV的疫苗。综上所述，研发大鼠HEV的ORF2的重组蛋白对大鼠HEV的检测手段和疫苗制备等防治策略至关重要。
发明内容
有鉴于此，本发明的目的在于提供一种正戊型肝病毒属基因C1型病毒ORF2 重组蛋白及其制备方法和应用，所述大鼠HEV-C1 ORF2重组蛋白，是根据已知的大鼠HEV的ORF2所设计的一致性氨基酸序列，能够最大限度地保留大鼠 HEV的免疫原性；所述重组蛋白能够刺激不同物种动物产生特异性抗体。
本发明提供了一种大鼠HEV-C1 ORF2重组蛋白，其氨基酸序列如SEQ ID No.1所示；
或者其氨基酸序列与SEQ ID No.1所示序列具有至少90％的同一性。
本发明提供了编码所述的大鼠HEV-C1 ORF2重组蛋白的基因，所述基因的核苷酸序列如SEQ ID No.2所示。
本发明还提供了一种大鼠HEV-C1 ORF2重组蛋白的表达载体，包括初始载体和所述的基因。
优选的，所述基因插入到所述初始载体的多克隆位点。
本发明还提供了一种表达所述的大鼠HEV-C1 ORF2重组蛋白的重组菌株，所述重组菌株转化有所述的表达载体。
优选的，所述重组菌株以大肠杆菌BL21为宿主菌。
本发明提供了所述的大鼠HEV-C1 ORF2重组蛋白在制备预防人感染大鼠HEV的疫苗中的应用。
优选的，所述疫苗为亚单位疫苗。
本发明还提供了所述的大鼠HEV-C1 ORF2重组蛋白在制备检测人感染大鼠 HEV的试剂中的应用。
本发明提供了所述的大鼠HEV-C1 ORF2重组蛋白免疫动物后获得的单克隆抗体和/或多克隆抗体。
本发明提供了所述的单克隆抗体和/或多克隆抗体在制备检测大鼠HEV的试剂中的应用。
本发明提供了所述的单克隆抗体和/或多克隆抗体在制备预防人感染大鼠 HEV的疫苗中的应用。
与现有技术相比，本发明具有如下有益效果：本发明提供的正戊型肝病毒属基因C1型病毒ORF2重组蛋白是根据已知的大鼠HEV的ORF2所设计的一致性氨基酸序列，能够最大限度地保留大鼠HEV的免疫原性；所述重组蛋白能够刺激不同物种动物产生特异性抗体。通过ELISA实验，所述重组蛋白能够检测不同物种(兔、大鼠和小鼠)中免疫大鼠HEV-ORF2后的抗体水平，可以有效地区别HEV-A(human HEV，hHEV)ORF2和HEV-C1 ORF2免疫后其抗原抗体相互作用的差异。免疫HEV-C1 ORF2蛋白的大鼠血清中产生的针对HEV-C1 ORF2的特异性抗体能有效阻断HEV-C1 ORF2病毒样颗粒与大鼠肝癌细胞 RH-35的特异性结合，而未免疫大鼠血清、免疫HEV-A ORF2蛋白的大鼠血清无阻断效果，证实所述重组蛋白能够用于制备HEV-C1疫苗。
附图说明
图1为HEV-C1 ORF2氨基酸序列同源性比对及一致性序列的生成；
图2为HEV-C1 ORF2亲缘关系图；
图3为pET-21a(+)-Rat ORF2357-595质粒图谱；
图4为HEV-C1 ORF2 PCR产物；
图5为利用SDS-PAGE考马斯染色对HEV-C1 ORF2的纯化进行鉴定，从左至右泳道中加样分别为Marker、过柱前样品、过柱后样品、10ml wash buffer洗脱、5ml elutionbuffer洗脱样品、透析前样品、透析后样品；
图6为利用Western blot鉴定变性与非变性HEV-C1 ORF2重组蛋白；
图7为电镜观察HEV-C1 ORF2重组蛋白能够形成病毒样颗粒；
图8为HEV-C1 ORF2免疫小鼠、新西兰大白兔和大鼠的抗体水平，其中A、 B和C分别为小鼠、新西兰大白兔和大鼠；
图9为HEV-C1 ORF2重组蛋白免疫产生的抗体特异地与HEV-C1 ORF2、HEV-A ORF2和HEV-A ORF3相互作用结果，其中A为大鼠1，B为大鼠2；
图10为HEV感染病人血清与HEV-A ORF2和HEV-C1 ORF2之间的相互作用分析；
图11为利用HEV-C1 ORF2蛋白免疫兔子制备的多抗用于HEV-C1 ORF2蛋白检测的结果；
图12为HEV-C1 ORF2能形成病毒样颗粒，并能与大鼠肝癌细胞RH-35的细胞膜发生特异性结合，可充分模拟大鼠HEV病毒吸附在易感细胞表面的过程，其中A为对照与大鼠肝癌细胞RH-35表面结合情况，B为HEV-C1 ORF2与大鼠肝癌细胞RH-35表面结合情况；
图13为免疫HEV-C1 ORF2蛋白的大鼠血清中产生的针对HEV-C1 ORF2的特异性抗体能有效阻断HEV-C1 ORF2病毒样颗粒与RH-35细胞的特异性结合，而未免疫大鼠血清、免疫HEV-A ORF2蛋白的大鼠血清无阻断效果；其中A为阴性对照组，RH-35细胞未与rHEV-ORF2共同孵育，仅加入抗hHEV-ORF2、 rHEV-ORF2的免疫荧光图，B为RH-35细胞分别与rHEV-ORF2、rHEV-ORF2 与抗hHEV-ORF2、rHEV-ORF2与抗rHEV-ORF2相互作用的免疫荧光图。
具体实施方式
本发明提供了一种大鼠HEV-C1 ORF2重组蛋白，其氨基酸序列如SEQ ID No.1所示，具体如下：
MIVQVLFNIADTLLGGLPTDLVSNAGGQLFYGRPQVSENGEPSVKLYTS VEAAQLDQGVTIPHDIDLGLSAVTLQDFDNQHLQDRPTPSPAPARPITNWRSG DVVWVTLPSAEYAQSQSAMGSHPAYWSEEATIINVATGQRASVSSIKWDQV TLNGKALHKETHSGLVYYQLPLMGKISFWQQGTTKAGYTYNYNTTDSDSLW VWWDGASKAYLYLSTYTTMLGAGPVNITGLGAIGPS。
本发明提供的HEV-C1 ORF2重组蛋白由240个氨基酸构成，分子量为 26kDa。
在本发明中，所述HEV-C1 ORF2重组蛋白的氨基酸序列是通过将NCBI数据库中的HEV-C1 ORF2的氨基酸序列进行对比后，并通过Vector NTI Advance 11.5AlignX功能分析构建获得的一致性序列。
本发明还提供了一种大鼠HEV-C1 ORF2重组蛋白，其氨基酸序列与SEQ ID No.1所示序列具有至少90％的同一性，优选的具有至少95％的同一性。在本发明中，SEQ ID No.1所示序列为人工分析构建的一致性序列，利用的是一致性序列的特点，因此与SEQ ID No.1所示序列具有至少90％同一性的序列具有与SEQ ID No.1所示序列相当的性能，能够获得与SEQ ID No.1所示序列相当的试验效果。
序列同一性通常以百分比同一性(或相似性或同源性)来测量；百分比越高，两个序列越相似。用于比对比较序列的方法是本领域熟知的。各种程序和比对算法描述如下：Smith T,Waterman M.Comparison of biosequences.Advances in AppliedMathematics.1981Dec,2(4):482-489；Needleman S,Wunsch CA general methodapplicable to the search for similarities in the amino acid sequence of twoproteins,J Mol Biol.1970Mar；48(3):443-53；Pearson W,Lipman D.Improved toolsfor biological sequence comparison.Proc Natl Acad Sci U S A.1988 Apr；85(8):2444-8；Higgins D,Sharp P.CLUSTAL:apackage for performing multiple sequencealignment on a microcomputer.Gene.1988Dec 15；73(1):237-44；Higgins D, SharpP.Fast and sensitive multiple sequence alignments on a microcomputer. ComputAppl Biosci.1989Apr；5(2):151-3；Corpet F.Multiple sequence alignment withhierarchical clustering.Nucleic Acids Res.1988Nov 25；16(22):10881–10890；Huang X,Miller W,Schwartz S,Hardison R.Parallelization of a local similarityalgorithm.Comput Appl Biosci.1992Apr；8(2):155-65；Pearson W.Using the FASTAprogram to search protein and DNA sequence databases.Methods Mol Biol. 1994；24:307-31；Altschul S,Boguski M,Gish W,Wootton J.Issues in searching molecularsequence databases.Nat Genet.1994Feb；6(2):119-29.以上文献内容提出了序列比对方法和同源性计算的详细考量。
举例而言，比对工具ALIGN(Myers E and Miller W.Optimal Alignments inLinear Space.CABIOS 4,1988,11-17.)或LFASTA(Pearson W,Lipman D. Improved toolsfor biological sequence comparison.Proc Natl Acad Sci U S A.1988 Apr；85(8):2444-8.)可用于进行序列比较(Internet 1996,W.R.Pearson and the University ofVirginia，fasta版本2.0，发布日期1996年12月)。ALIGN将整个序列进行相互比较，而LFASTA则比较局部区域的相似性。例如，这些比对工具及其各自的教程可在因特网上的国家超级计算机应用中心(National Center for Supercomputer Applications，简称NCSA)网站上获得。或者，为了比较大于约 30个氨基酸的氨基酸序列，可以使用设置为默认参数的默认BLOSUM62矩阵执行Blast 2序列功能(空位存在罚分为11，每个残基缺口罚分为1)。当比对短肽(少于约30个氨基酸)时，应使用设置为默认参数的PAM30矩阵，采用Blast 2序列功能进行比对(开放空位9，延伸空位1罚分)。BLAST序列比较系统可从例如 NCBI网站获得。(Altschul S,Gish W,Miller W,Myers E,Lipman D.Basic local alignment searchtool.J Mol Biol.1990Oct 5；215(3):403-10；Gish W,States D. Identification ofprotein coding regions by database similarity search.Nat Genet. 1993Mar；3(3):266-72；Madden T,Tatusov R,Zhang J.Applications of network BLASTserver.Methods Enzymol.1996；266:131-41；Altschul S,Madden T,
A,ZhangJ,Zhang Z,Miller W,Lipman D.Gapped BLAST and PSI-BLAST:a new generation ofprotein database search programs.Nucleic Acids Res.1997Sep 1；25(17):3389-402；Zhang J,Madden T.PowerBLAST:a new network BLAST application for interactiveor automated sequence analysis and annotation.Genome Res.1997Jun；7(6):649-56.)
本发明提供了编码所述的大鼠HEV-C1 ORF2重组蛋白的基因，所述基因的核苷酸序列如SEQ ID No.2所示，具体如下：
atgattgtgcaagtgctgtttaacattgcagataccctgctgggtggcctgccaactgatctggtgagcaatgctggtg gtcagttgttttatggcagaccacaagtgagtgaaaatggtgaaccaagtgtgaaactgtatactagtgtggaagcagcaca gctggatcaaggtgtgaccattccacatgatattgatctgggtctgagtgcagtgaccctgcaagattttgataatcagcatct gcaagatagacctaccccaagccctgccccggcgagaccaattaccaactggagaagtggtgatgtagtttgggtgaccc tgccaagtgcagaatatgcacagagtcagagtgccatgggcagccatcctgcctattggagtgaagaagccaccattatta atgtggccactggtcagagagcaagtgtgagcagcattaaatgggatcaagtgaccctgaatggcaaagccctgcataaa gaaacccatagtggcctggtgtattatcagctgccactgatgggcaaaattagcttttggcagcaaggcaccaccaaagctg gctatacctataactataacaccactgatagtgatagcctgtgggtttggtgggatggtgcaagcaaagcctatctgtatctga gcacctataccactatgctgggtgctggccctgtcaacattactggcctgggtgccattggaccaagc。
本发明还提供了一种大鼠HEV-C1 ORF2重组蛋白的表达载体，包括初始载体和所述的基因。本发明对具体的初始载体的类型并没有特殊限定，采用常规市售的商业化表达载体，例如真核表达质粒pcDNA3.1等表达质粒、慢病毒/腺病毒载体的相关表达质粒，如pWPI，pAAV等均可。在本发明的一个具体实施过程中，所述初始载体优选为pET-21a(+)。在本发明中，所述基因优选的插入到初始载体的Nde I和Xho I酶切位点之间。本发明对所述鼠HEV-C1 ORF2重组蛋白的表达载体的构建方法没有特殊限定，采用本领域常规的重组载体的构建方法即可。在本发明具体实施过程中，采用双酶切和连接的方法构建重组表达载体。
本发明还提供了一种表达所述的大鼠HEV-C1 ORF2重组蛋白的重组菌株，所述重组菌株转化有所述的表达载体。
在本发明中，所述重组菌株优选的以大肠杆菌BL21为宿主菌；本发明对所述转化的方法没有特殊限定，采用本领域常规的表达载体转化方法即可。
本发明提供了所述的大鼠HEV-C1 ORF2重组蛋白在制备预防人感染大鼠 HEV的疫苗中的应用。在本发明中，所述疫苗优选为亚单位疫苗。在本发明中，所述疫苗中包括大鼠HEV-C1 ORF2重组蛋白和弗氏完全佐剂；所述疫苗中大鼠 HEV-C1 ORF2重组蛋白的浓度优选为0.8～1.2mg/mL，进一步优选为1mg/mL。在本发明中，用所述疫苗免疫大鼠、小鼠、新西兰大白兔均能够得到较高效价的抗体。
本发明还提供了所述的大鼠HEV-C1 ORF2重组蛋白在制备检测人感染大鼠 HEV的试剂中的应用。本发明研究发现HEV-A感染病人产生的抗体特异地与 HEV-A ORF2相互作用，而不与所述大鼠HEV-C1 ORF2重组蛋白相互作用。以大鼠HEV-C1 ORF2重组蛋白不能够检测HEV-A戊型肝炎病毒感染的病人，该大鼠HEV-C1 ORF2重组蛋白可以用来建立ELISA方法实现对人感染HEV-C1型病毒的特异性诊断。
本发明提供了所述的大鼠HEV-C1 ORF2重组蛋白免疫动物后获得的单克隆抗体和/或多克隆抗体。本发明对所述单克隆抗体和/或多克隆抗体的分离纯化方法没有特殊限定，采用本领域常规的单克隆抗体和/或多克隆抗体的分离纯化方法即可。
本发明还提供了所述的单克隆抗体和/或多克隆抗体在制备检测大鼠HEV的试剂中的应用。
本发明还提供了所述的单克隆抗体和/或多克隆抗体在制备预防人感染大鼠 HEV的疫苗中的应用。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
HEV-C1 ORF2氨基酸序列的比对及ORF2一致性氨基酸序列的设计
将戊型肝炎病毒HEV-C1 ORF2的序列从NCBI下载，序列号分别为： HEV-C1HEV(AB847305)，HEV-C1 HEV(LC145325)，HEV-C1 HEV(AB847306)， HEV-C1 HEV(AB847309)，HEV-C1 HEV(AB847308)，HEV-C1 HEV (GU345042)，HEV-C1 HEV(GU345043)，HEV-C1 HEV(JN167537)，HEV-C1 HEV (KM516906)，HEV-C1 HEV(JN167538)，HEV-C1 HEV(MK050105)，HEV-C1 HEV (LC225389)，HEV-C1 HEV(AB847307)，HEV-C1 HEV(JX120573)，HEV-C1 HEV(MG813927)，HEV-C1 HEV(LC225388)。
将下载的所有导入到VectorNTI(11.5.3)，利用Alignment功能将所有序列进行比对(结果如图1所示)，并利用Guide Tree功能对其进行亲缘关系分析(结果如图2所示)，通过Consensus Calculation分析构建ORF2的一致性序列SEQ ID No.1。
SEQ ID No.1：
MIVQVLFNIADTLLGGLPTDLVSNAGGQLFYGRPQVSENGEPSVKLYTSV EAAQLDQGVTIPHDIDLGLSAVTLQDFDNQHLQDRPTPSPAPARPITNWRSGD VVWVTLPSAEYAQSQSAMGSHPAYWSEEATIINVATGQRASVSSIKWDQVTLN GKALHKETHSGLVYYQLPLMGKISFWQQGTTKAGYTYNYNTTDSDSLWVW WDGASKAYLYLSTYTTMLGAGPVNITGLGAIGPS。
实施例2
HEV-C1 ORF2表达质粒的构建
根据ORF2序列比对获得的一致性的氨基酸序列设计核苷酸序列，并分别根据大肠杆菌宿主进行密码子优化，通过基因合成获得编码ORF2序列的基因。利用PCR扩增该段基因，在引物两端加入NdeI和XhoI酶切位点，将编码截短后的ORF2(357-595aa)片段的基因和pET21a载体用进行NdeI和XhoI双酶切和胶回收，然后利用T4连接酶进行连接，构建成重组表达质粒pET-21a(+)-Rat ORF2357-595(质粒图谱如图3所示)。该重组表达质粒带有起始密码子和His 标签，使得目的蛋白与His标签重组表达。
具体步骤如下：
人工合成HEV-C1 ORF2(357-595aa)一致性序列的基因，该基因片段长为 720bp，基因序列如SEQ ID No.2所示，该片段由苏州金唯智生物科技有限公司合成，具体如下：
atgattgtgcaagtgctgtttaacattgcagataccctgctgggtggcctgccaactgatctggtgagcaatgctggtg gtcagttgttttatggcagaccacaagtgagtgaaaatggtgaaccaagtgtgaaactgtatactagtgtggaagcagcaca gctggatcaaggtgtgaccattccacatgatattgatctgggtctgagtgcagtgaccctgcaagattttgataatcagcatct gcaagatagacctaccccaagccctgccccggcgagaccaattaccaactggagaagtggtgatgtagtttgggtgaccc tgccaagtgcagaatatgcacagagtcagagtgccatgggcagccatcctgcctattggagtgaagaagccaccattatta atgtggccactggtcagagagcaagtgtgagcagcattaaatgggatcaagtgaccctgaatggcaaagccctgcataaa gaaacccatagtggcctggtgtattatcagctgccactgatgggcaaaattagcttttggcagcaaggcaccaccaaagctg gctatacctataactataacaccactgatagtgatagcctgtgggtttggtgggatggtgcaagcaaagcctatctgtatctga gcacctataccactatgctgggtgctggccctgtcaacattactggcctgggtgccattggaccaagc
利用ORF2 PCR引物进行扩增，基因片段长730bp，使其在5’端和3’端分别加入NdeI和XhoI酶切位点，PCR扩增引物如下：
正向引物HEV-C1 ORF2-F：
5’-ggaattccatatgattgtgcaagtgctgtttaac-3’(SEQ ID No.3，Tm＝61℃)。
反向引物HEV-C1 ORF2-R：
5’-ccgctcgaggcttggtccaatggcacccag-3’(SEQ ID No.4)。
PCR反应体系为如表1。
表1PCR反应体系
利用琼脂糖电泳对PCR产物大小进行鉴定，结果如图4所示，序列长度符合预期。
利用Nde I和Xho I对目的片段和带有His标签的载体pET-21a(+)分别进行酶切(酶切体系如表2)，再用T4连接酶(连接体系如表3)将处理后的两者进行过夜连接。
表2利用Nde I和Xho I对目的片段和载体进行酶切
| 组成 | 25μL体系用量 |
| DNA(PCR/plasmids) | 0.5μg |
| 10X rCutSmart Buffer | 2.5μL(1X) |
| NdeI | 0.5μL(20units) |
| XhoI | 0.5μL(20units) |
| Nuclease-fre eWater | to25μL |
(1)目的片段(730bp)与载体片段物质的量之比为5:1，据此计算出目的片段所需的量，见表3。
(2)将样品置于连接仪内，16℃，连接过夜。
表3连接体系
| 组分 | 体积 |
| T4 DNA Ligase | 1μL |
| 10x T4 DNA Ligase Buffer | 2μL |
| 10x Buffer | 2μL |
| 目的片段 | 55ng |
| 载体片段 | 80ng |
| 灭菌蒸馏水 | 补足 |
| 总体积 | 20μL |
转化(DH5α)
(1)取大肠杆菌DH5α感受态细胞50μL菌体使其迅速在冰上融化，加入连接产物10μL，轻轻旋转混合，冰浴30min。
(2)立即置入42℃水浴锅内，热休克90s，勿晃动，使其达到准确热激温度。
(3)迅速转移至冰上，冰浴2min。
(4)在超净台中加650μL LB培养基至样品管内轻轻混匀。
(5)将样品管置于摇床中，37℃，220rpm，振荡培养1h。
(6)5000rpm，室温，离心1min。
(7)弃上清550μL，余约150μL混匀，全部加至抗生素平板上并涂板，至平板无液体流动，正放10min，倒放平板37℃培养14h。挑取多个菌落克隆，利用加入氨苄的LB培养基对其进行培养，利用天根质粒小提试剂盒(DP103)提取质粒，送至苏州金唯智生物科技有限公司进行测序鉴定，结果正确为 pET-HEV-C1-ORF2-His。
实施例3
HEV-C1 ORF2重组蛋白的表达与纯化
将重组表达质粒pET-21a(+)-Rat ORF2357-595转化到大肠杆菌表达宿主菌 BL21中，加入异丙基-β-D-硫代吡喃和半乳糖苷诱导表达，最后收集菌液，超声裂解离心后，对其进行纯化，并利用SDS-PAGE和Westernblot鉴定蛋白的表达，其具体步骤如下：
(1)用50ml离心管收集菌体(4000rpm，离心10min收集沉淀)，多次离心之后，将菌体沉淀收至1个50ml离心管中。放入-20℃冻存。
(2)取出冻存的菌液，每管用5mlPBS+50μl蛋白酶抑制剂+50μl溶菌酶(溶菌酶的终浓度为1mg/ml)，充分混匀，静置30min。
(3)超声：移入10ml离心管，超声45min。
(4)离心：11000rpm，4℃，30min，弃上清。
(5)用5ml包涵体洗涤液(表4)匀浆洗涤三次，4℃，12000rpm离心10min 收集沉淀。
(6)用5ml包涵体变性液(表5)溶解沉淀，4℃，12000rpm离心10min 收集上清。
表4包涵体变性液配方
表5包涵体复性液的配制
(7)用4ml去离子水冲洗出储存缓冲液，使用至少5ml的Lysis Buffer平衡柱子；
(8)将蛋白样品加入柱中，让蛋白吸附在柱子中；
(9)用15ml Wash Buffer冲洗柱子；
(10)用Elution Buffer洗脱，用5ml Elution buffer洗脱目的蛋白，将洗脱的蛋白用EP管收集。
(11)利用SDS-PAGE进行考马斯亮蓝染色对纯化效率进行鉴定。其操作步骤如下：
①总蛋白的提取：加入相应量的5×蛋白上样缓冲液，混匀后煮样10min，冷却后，样品收入-20℃储存。
②SDS-PAGE电泳：配制12.5％分离胶和4％浓缩胶，先配制分离胶，充分混匀后灌入凝胶板中，用无水乙醇作为压线液，等待40min，倾倒弃去上层无水乙醇并用吸水纸吸去残余液体，再配制浓缩胶，充分混匀后灌注上层，插入成型梳后，室温静置约40min，胶完全凝固后收入4℃冰箱，储存备用。配制电泳液见表6，将凝胶板放入电泳架，加入电泳液后拔去成型梳，每个凝胶孔加入15μL 蛋白样品，其中一孔，加入蛋白Marker。将电泳架放入电泳槽，补足内槽外槽电泳液后开始电泳，70V电泳压线约30min，待样品电泳至浓缩胶与分离胶分界线时更改电压至120V，待溴酚蓝跑至分离胶底部时停止电泳。
表6电泳液配方
考马斯亮蓝染色SDS-PAGE结果如图5显示，ORF2能够表达，且与Ni-NTA 柱子有良好的结合，通过Elution buffer能够正常洗脱。Western blot结果如图6 显示，非变性的ORF2形成二聚体，蛋白变性后，二聚体变为单体。
(12)利用His标签抗体(1:5000倍稀释)进行Western blot，ORF2蛋白对变性和非变性进行鉴定，其操作步骤如下
蛋白提取与SDS-PAGE如步骤(11)所示，在此基础上，裁剪与目的胶块大小相同的PVDF膜，放入甲醇中浸泡30s，使PVDF膜充分激活，再将PVDF 膜放入回收利用的湿转液中，浸泡3～5min。裁剪与胶块大小相同的白色滤垫，充分浸泡后放在湿转夹上，转膜在湿转电转仪中进行，排列顺序为：白板-海绵垫-白色滤纸-PVDF膜-胶-白色滤纸-海绵垫-黑板。接通电源，100V稳压，湿转 100min。将膜浸泡于封闭液中(5％脱脂奶粉)，室温摇床封闭2h。封闭结束后，将PVDF膜进行简单清洗后裁剪与目的蛋白大小一致，放入抗体孵育盒中，将His抗体血清利用1％BSA分别稀释至5000倍，加入到孵育盒中。置于4℃摇床孵育过夜。第二天取出抗体孵育盒，室温摇床复温30min后吸取一抗，用 TBST洗涤缓冲液震荡洗涤3次，每次10min。将PVDF膜置于抗体孵育盒中，加入稀释好的抗兔IgG抗体，稀释比例为1:5000(稀释液为1％BSA)，室温摇床孵育2h，吸取二抗，用TBST洗涤缓冲液震荡洗涤3次，每次10min。利用 ECL对Western blot进行显色。
表7Lysis Buffer的配制
表8Wash Buffer的配制
表9Elution Buffer的配制
实施例4
HEV-C1 ORF2重组蛋白形成类病毒样颗粒，能够模拟HEV-C1病毒结构，免疫原性强，是HEV-C1亚单位疫苗候选株
将HEV-C1 ORF2重组蛋白2mg/ml溶液分别取原液100μl(1滴的量)，滴加在装有支持膜的铜网上，静置2min，用吸水纸从铜网边缘吸去多余液体，滴加100μl(1滴的量)1.5％醋酸铀(pH＝7)，静置2min，用吸水纸从铜网边缘吸去多余液体，晾干2min，利用电镜进行观察。
观察结果如图7所示，发现该HEV-C1 ORF2重组蛋白能够形成病毒样颗粒，大小为40～60nm，该病毒样颗粒能够模拟HEV-C1病毒粒子的衣壳结构，具有与病毒粒子同等的免疫原性，是HEV-C1亚单位疫苗的候选株。
实施例5
利用HEV-C1 ORF2重组蛋白建立血清ELISA方法，并利用该蛋白免疫动物后其血清抗体效价的鉴定
1.利用HEV-C1 ORF2重组蛋白免疫大鼠、小鼠和新西兰大白兔，并在不同时间段采集血清
1.1免疫大鼠：免疫前用毛细吸管用眼内眦取血的方式获得大鼠血液500μl，分离血清做为阴性对照。将纯化好的HEV-C1 ORF2重组蛋白稀释至100μL (200μg)与等体积弗氏完全佐剂乳化，在大鼠背部多部位喷洒酒精消毒后皮下注射免疫。免疫两周后再次麻醉大鼠，获取一免后血清500μl，检测其效价。两周后进行第二次免疫。实验共需免疫大鼠六次，最后一次免疫两周后经心脏采血获取大鼠全部血液并将其处死，检测六免后血清效价。
1.2免疫小鼠：免疫前用毛细吸管用眼内眦取血的方式获得小鼠血液200μl，分离血清做为阴性对照。将纯化好的HEV-C1 ORF2重组蛋白稀释至50μL (100μg)与等体积弗氏完全佐剂乳化皮下注射免疫。免疫一周后小鼠眼内眦取血获得一免小鼠血清200μl，检测血清效价。免疫两周进行第二次免疫，二免两周后进行第三次免疫。实验共需免疫小鼠六次，最后一次免疫一周后眼球取血获取小鼠全部血液，后将小鼠脱颈处死，检测六免后血清效价。
1.3免疫新西兰大白兔：注射免疫前从兔子耳缘静脉采血2ml，分离血清当做阴性对照。将纯化好的HEV-C1 ORF2重组蛋白(1mg)用生理盐水稀释至1ml 与等体积1ml的弗氏完全佐剂乳化后进行免疫，2周后进行第二次免疫。实验共需要注射免疫5次，每次免疫前都要耳缘静脉采血检测上一次免疫后产生抗体的效价，最后一次免疫一周后耳缘静脉采血检测效价，之后用10ml 10％水合氯醛麻醉兔子，心脏采血采集兔子全部血液。
2.建立ELISA方法，测定不同种动物体内抗ORF2的抗体水平
①利用200ng的HEV-C1 ORF2重组蛋白包被ELISA板，并利用同样带有 His-tag的ORF3作为阴性对照进行包板；
②将采集的HEV-C1 ORF2重组蛋白免疫的动物血清从1:1000开始进行横向的倍比稀释，然后与ELISA板上的HEV-C1 ORF2重组蛋白在37℃孵育1小时，弃掉孵育血清，用PBST洗三次；
③加入抗不同动物血清(抗大鼠、抗小鼠和抗兔)的IgG-HRP二抗(1:500 倍稀释)，在37℃孵育1小时，弃掉二抗，用PBST洗三次；
④加入TMB显色液进行显色2min，并加入H2SO4进行终止，利用ELISA 分光光度计读板机在450nm对其结果进行检测。
所用试剂：
①包被液：pH9.60.05M碳酸缓冲液
②终止液：1N硫酸
③封闭液：5％(v/v)牛奶
④ELISA洗液：PBS+0.05％Tween20。
检测结果显示，小鼠6次免疫HEV-C1 ORF2重组蛋白后，其抗体效价达到很高的滴度，抗体稀释1000倍到32000倍，仍然有很高的抗体效价(图8中的 A)；新西兰大白兔免疫后，将血清稀释16000倍仍处于ELISA反应的平台期，稀释128000倍仍能与HEV-C1 ORF2重组蛋白有很好的相互作用(图8中的B)；大鼠5次免疫后，将其血清稀释4000倍后，仍能与ORF2蛋白产生相互作用。
以上结果说明，利用该抗原包被的ELISA方法，能够检测到不同物种免疫后的HEV-C1 ORF2血清抗体，并且检测结果呈抗体浓度依赖性，因此利用该 HEV-C1 ORF2重组蛋白作为抗原可以建立ELISA方法用于诊断HEV-C1感染病人；并且以上不同动物的免疫实验说明，该HEV-C1 ORF2蛋白有非常好的免疫原性。
实施例6
HEV-C1 ORF2重组蛋白免疫大鼠产生的抗体对HEV-C1 ORF2和HEV-A ORF2交叉免疫性
分别利用HEV-C1 ORF2重组蛋白和HEV-A ORF2以及HEV-A ORF3的蛋白包被ELISA板，用HEV-C1 ORF2重组蛋白五次免疫大鼠后的血清倍比稀释与三种不同的抗原进行孵育，其方法如下：
(1)利用200ng的抗原蛋白(HEV-C1 ORF2、HEV-A ORF2和HEV-A ORF3) 包被ELISA板；
(2)将分别采集HEV-C1 ORF2重组蛋白免疫的两只大鼠血清从1:1000开始进行横向的倍比稀释，然后与ELISA板上的抗原蛋白在37℃孵育1小时，弃掉孵育血清，用PBST洗三次；
(3)加入抗大鼠IgG-HRP二抗(1:500倍稀释)，在37℃孵育1小时，弃掉二抗，用PBST洗三次；
(4)加入TMB显色液进行显色2min，并加入H2SO4进行终止，利用ELISA 分光光度计读板机在450nm对其结果进行检测。
对两只大鼠免疫后血清效价进行鉴定发现，HEV-C1 ORF2重组蛋白免疫后产生的抗体能特异地与HEV-C1 ORF2相互作用，而与HEV-A ORF2和HEV-A ORF3的蛋白没有相互作用。以上结果说明，HEV-A ORF2蛋白与HEV-C1免疫抗体交叉免疫反应水平很低，因此利用HEV-A ORF2建立的ELISA检测方法不能用于大鼠HEV感染的诊断，因此利用HEV-C1建立的诊断试剂盒至关重要。
实施例7
测定HEV-A病毒感染患者血清与HEV-C1 ORF2重组蛋白之间的交叉免疫反应
分别利用HEV-C1 ORF2重组蛋白和HEV-A ORF2的蛋白包被ELISA板，用HEV-A感染病人的血清倍比稀释与两种不同的抗原进行孵育，实验过程如下：
(1)利用200ng的HEV-C1 ORF2重组蛋白和HEV-A ORF2包被ELISA板；
(2)将戊型肝炎(HEV-A)患者的血清从1:100倍开始进行倍比稀释，加入100μL稀释后抗体与ELISA板上的抗原蛋白在37℃孵育1小时，弃掉孵育血清，用PBST洗三次；
(3)加入抗大鼠IgG-HRP二抗(1:1000倍稀释)，在37℃孵育1小时，弃掉二抗，用PBST洗三次；
(4)加入TMB显色液进行显色2min，并加入H2SO4进行终止，利用ELISA 分光光度计读板机在450nm对其结果进行检测。
对其结果进行鉴定发现，HEV-A感染病人产生的抗体特异地与HEV-A ORF2 相互作用，而不与HEV-C1 ORF2重组蛋白相互作用。该实验证明，以HEV-C1 ORF2重组蛋白不能够检测HEV-A戊型肝炎病毒感染的病人，该蛋白可以用来建立ELISA方法对人感染HEV-C1型病毒进行诊断。
实施例8
HEV-C1 ORF2重组蛋白免疫产生的多克隆抗体可以作为实验检测HEV-C1 手段。
ORF2基因序列长度为：730bp，以rHEV毒株LCK-3110(GenBank: MG813927.1)为模板，以pcDNA3.1-Flag为载体，以在其上、下游分别添加Xho I和EcoRI酶切位点，设计用于扩增基因序列的引物。
上游引物：5’-aacctcgagatgtctgtcgttgtcgtgctcg-3’(SEQ ID No.5)
下游引物：5’-aacgaattcgacactgtcggctgctacggct-3’(SEQ ID No.6)
PCR扩增ORF2全长片段
(1)在0.2mL EP管中加入以下反应混合物，反应体系如表10。
表10PCR反应体系
(2)将样品管置于PCR仪器中，反应程序见表11，循环数为35，ORF2 蛋白PCR结果。
表11PCR扩增反应程序
| 温度 | 时间 |
| 预变性98℃ | 5min |
| 变性98℃ | 10s |
| 退火65℃ | 30s |
| 延伸72℃ | 40s |
PCR产物的胶回收
(1)PCR产物经2％琼脂糖凝胶电泳后，在紫外灯下小心把目的胶块切下。
(2)将高压后的空1.5mL EP管进行称量并记录重量后，取目的胶块收入 EP管中，再次称重，两次重量的差为目的胶块的重量。
(3)根据1μg胶+1μL Binding Buffer的要求加入正确量的Binding Buffer 在EP管内。
(4)将其放入55℃水浴锅内，水浴10min后，取出EP管，仔细观察胶块是否完全溶解。若未完全溶解，可再次短暂加热，直至完全溶解。
(5)将DNA琼脂糖溶液转移至吸附柱内，静置2min，离心12000rpm， 75s。(吸附柱每次仅能容纳700μL溶液，若单次不能全部加入吸附柱中，余下部分可待离心后加入吸附柱内)
(6)弃废液，向吸附柱内加入700μL Washing Buffer，离心10000rpm，75 s。
(7)重复上一步。
(8)弃废液，空转10000rpm，2min。
(9)将柱子转移到一个新的高压后的1.5mL EP管内，打开盖子，静置2min。
(10)向柱子的中心加入30μL Elution Buffer，静置2min后，离心13000rpm，1min。
(11)测定回收产物的浓度，并记录在EP管管壁，储存于-20℃。
目的片段和载体的酶切
(1)在0.2mL EP管中加入以下反应混合物。
表12酶切体系
| 组分 | 体积 |
| 酶1 | 1μL |
| 酶2 | 1μL |
| 10x Buffer | 2μL |
| DNA | ≤1μg |
| 灭菌蒸馏水 | 补足 |
| Total volume | 20μL |
(2)按照反应体系混匀样品后，短暂瞬离。放入37℃培养箱，酶切3h。
目的片段与载体片段的连接
(1)目的片段(730bp)与载体片段(5500bp)物质的量之比为3:1，据此计算出目的片段所需的量，见表13。
(2)将样品置于连接仪内，16℃，连接过夜。
表13连接体系
| 组分 | 体积 |
| T4 DNA Ligase | 1μL |
| 10x T4 DNA Ligase Buffer | 2μL |
| 10x Buffer | 2μL |
| 目的片段 | 32ng |
| 载体片段 | 80ng |
| 灭菌蒸馏水 | 补足 |
| Total volume | 20μL |
转化大肠杆菌(DH5α)
(1)取大肠杆菌DH5α感受态细胞50μL菌体使其迅速在冰上融化，加入连接产物10μL，轻轻旋转混合，冰浴30min。
(2)立即置入42℃水浴锅内，热休克90s，勿晃动，使其达到准确热激温度。
(3)迅速转移至冰上，冰浴2min。
(4)在超净台中加650μL LB培养基至样品管内轻轻混匀。
(5)将样品管置于摇床中，37℃，220rpm，振荡培养1h。
(6)5000rpm，室温，离心1min。
(7)弃上清550μL，余约150μL混匀，全部加至抗生素平板上并涂板，至平板无液体流动，正放10min，倒放平板37℃培养14h。
将重组质粒送测序进行鉴定，结果正确为pcDNA3.1-ORF2-FLAG.
2.利用HEV-C1 ORF2重组蛋白产生的多克隆抗体检测ORF2蛋白真核表达阳性细胞
将上述中构建的pcDNA3.1-ORF2-flag质粒转染HEK 293T细胞
(1)六孔板每孔接种6×105个细胞，加入2mL含10％胎牛血清的DMEM 高糖培养基，充分混匀后放入37℃，5％CO2细胞培养箱中培养，124h后，使细胞覆盖率达到80％。
(2)取4μg质粒和12μL脂质体PEI分别溶于100μL的DMEM高糖培养基，室温静置5min，将质粒溶液加入到脂质体溶液中，轻轻混匀后，室温静置 20min。
(3)向六孔板细胞中加入DNA-脂质体混合物200μL/孔，充分混匀后放入 37℃，5％CO2细胞培养箱中培养，5h后更换为含10％胎牛血清的DMEM高糖培养基，即完成细胞转染。
(4)加入4％多聚甲醛固定细胞，室温静置30min。
(5)弃掉多聚甲醛，沿侧壁加入PBS，洗2遍，洗掉剩余的多聚甲醛。
(6)加入0.2％Triton打孔，覆盖即可，室温静置15min。
(7)弃掉Triton，沿侧壁加入PBS洗两遍。
(8)向细胞孔中加入封闭液(5％脱脂奶粉)，室温静置60min。
(9)将HEV-C1 ORF2重组蛋白产生的多克隆抗体(兔抗体)以1:2500稀释和Flag抗体(鼠抗体)以1:5000稀释，共同在37℃孵育1小时。
(10)沿侧壁加入PBS，将孔板置于摇床上慢摇，洗3次，5min/次。
(11)避光加入再用Cora Lite 488(抗兔)/594(抗鼠)偶联的IgG(绿色/ 红色)(稀释度为1：500)和Hochest(蓝色)(稀释度为1：500)对细胞进行染色，37℃孵育2小时。
(12)沿侧壁加入PBS，将孔板置于摇床上慢摇，洗3次，5min/次。
(13)在倒置荧光显微镜下观察拍摄。
(14)结果如图11所示：免疫荧光实验中利用Rabbit anti-rHEV-ORF2多抗和Mouseanti-Flag抗体能特异性检测到含有Flag标签的rHEV-ORF2蛋白。
实施例9
HEV-C1 ORF2重组蛋白免疫产生的多克隆抗体能够阻断类病毒颗粒与细胞表面的结合，可以作为HEV-C1亚单位疫苗的主要成分。
1.HEV-C1 ORF2重组病毒样颗粒与RH-35细胞表面的吸附，其实验流程如下：
(1)将RH-35大鼠肝癌细胞(购于武汉普诺赛生物科技有限公司)按照2×105个/ml的密度在12孔板内铺板，37℃、5％CO2饱和度的培养箱中过夜培养。
(2)次日，细胞密度达到80％。将HEV-C1 ORF2纯化蛋白按照每孔终浓度20μg/mL与PBS共300μL在1.5mL EP管中混合，对照组为PBS，37℃孵育 1h。
(3)弃去孔板细胞上清，用预冷的PBS清洗细胞两次，每孔加入300μL 蛋白与PBS孵育混合液，对照孔加入300μL孵育PBS，孔板放置于冰上，摇床轻轻孵育30min。
(4)弃去孔板中的液体，用预冷的PBS清洗细胞，孔板置于冰上，摇床轻轻清洗3次，每次5min。
(5)每孔加入300μL 2％多聚甲醛(4％多聚甲醛由PBS稀释至2％)，孔板放置于冰上，摇床轻轻固定30min。
(6)重复(4)。
(7)1％Casein(PBST配制)作为封闭液，每孔加入300μL，常温封闭1h。
(8)弃去封闭液，用PBS清洗细胞，摇床清洗两次，每次5min。
(9)加入HEV-C1 ORF2兔多抗免疫血清，用1％BSA 1:3000稀释，常温孵育1h，用PBS清洗细胞三次。
(10)兔荧光二抗与Hoechst用1％BSA 1:500稀释，常温孵育1h，用PBS 清洗细胞三次。
(11)倒置荧光显微镜下观察细胞与蛋白的结合并拍照分析结果。
结果如图12所示，表明HEV-C1 ORF2重组蛋白形成的类病毒颗粒能够特异性结合到RH35细胞表面，能够模拟大鼠HEV与宿主细胞表面受体结合的生物学特性。
大鼠免疫血清的竞争结合试验
(1)将RH-35大鼠肝癌细胞按照1×104/孔的密度在96孔板内铺板，37℃、 5％CO2饱和度的培养箱中过夜培养。
(2)次日，细胞密度达到80％。将HEV-C1 ORF2、HEV-A ORF2纯化蛋白与大鼠免疫血清混合，分为三组：终浓度1:20PBS稀释的大鼠阴性血清+20μg/mL HEV-C1 ORF2蛋白；终浓度1:20PBS稀释的HEV-C1 ORF2大鼠免疫血清 +20μg/mL HEV-C1 ORF2蛋白；终浓度1:20PBS稀释的HEV-A ORF2大鼠免疫血清+20μg/mL HEV-C1 ORF2蛋白。37℃孵育1h。
(3)弃去孔板细胞上清，用预冷的PBS清洗细胞两次，每孔加入100μL相应蛋白与大鼠血清和PBS孵育混合液。孔板放置于冰上，在摇床上轻轻孵育30 min。
(4)弃去孔板中的液体，用预冷的PBS清洗细胞，孔板置于冰上，在摇床上慢摇清洗三次，每次5min。
(5)每孔加入100μL4％多聚甲醛，孔板放置于冰上，在摇床上慢摇固定 30min。
(6)重复(4)。
(7)1％Casein(PBST配制)作为封闭液，每孔加入100μL，常温封闭1h。
(8)弃去封闭液，用PBS清洗细胞，在摇床上清洗两次，每次5min。
(9)加入HEV-C1 ORF2兔免疫血清，1％BSA 1:3000稀释，常温孵育1h，用PBS清洗细胞三次。
(10)兔荧光二抗与Hoechst用1％BSA 1:500稀释，常温孵育1h，用PBS 清洗细胞三次。
(11)倒置荧光显微镜下观察加入大鼠免疫血清后，HEV-C1与HEV-A重组病毒样颗粒在RH-35大鼠肝癌细胞表面的吸附。
试验结果如图13所示，表明免疫HEV-C1 ORF2重组蛋白免疫的大鼠血清中产生的针对HEV-C1 ORF2的特异性抗体能有效阻断HEV-C1 ORF2病毒样颗粒与RH-35细胞的特异性结合，而未免疫大鼠血清、免疫HEV-A ORF2蛋白的大鼠血清无阻断效果。该实验证实免疫HEV-C1 ORF2重组蛋白免疫的大鼠血清中产生了具有保护活性的抗体，可阻断HEV-C1入侵宿主细胞，HEV-C1 ORF2 重组蛋白可作为一种有效的候选疫苗，而免疫HEV-A ORF2蛋白产生的抗体对大鼠HEV无交叉保护活性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 徐州医科大学
<120> 一种正戊型肝病毒属基因C1型病毒ORF2重组蛋白及其制备方法和应用
<160> 6
<170> SIPOSequenceListing 1.0
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<211> 240
<212> PRT
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Met Ile Val Gln Val Leu Phe Asn Ile Ala Asp Thr Leu Leu Gly Gly
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Leu Pro Thr Asp Leu Val Ser Asn Ala Gly Gly Gln Leu Phe Tyr Gly
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Arg Pro Gln Val Ser Glu Asn Gly Glu Pro Ser Val Lys Leu Tyr Thr
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Ser Val Glu Ala Ala Gln Leu Asp Gln Gly Val Thr Ile Pro His Asp
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Ile Asp Leu Gly Leu Ser Ala Val Thr Leu Gln Asp Phe Asp Asn Gln
65 70 75 80
His Leu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ala Arg Pro Ile
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Thr Asn Trp Arg Ser Gly Asp Val Val Trp Val Thr Leu Pro Ser Ala
100 105 110
Glu Tyr Ala Gln Ser Gln Ser Ala Met Gly Ser His Pro Ala Tyr Trp
115 120 125
Ser Glu Glu Ala Thr Ile Ile Asn Val Ala Thr Gly Gln Arg Ala Ser
130 135 140
Val Ser Ser Ile Lys Trp Asp Gln Val Thr Leu Asn Gly Lys Ala Leu
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His Lys Glu Thr His Ser Gly Leu Val Tyr Tyr Gln Leu Pro Leu Met
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Gly Lys Ile Ser Phe Trp Gln Gln Gly Thr Thr Lys Ala Gly Tyr Thr
180 185 190
Tyr Asn Tyr Asn Thr Thr Asp Ser Asp Ser Leu Trp Val Trp Trp Asp
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Gly Ala Ser Lys Ala Tyr Leu Tyr Leu Ser Thr Tyr Thr Thr Met Leu
210 215 220
Gly Ala Gly Pro Val Asn Ile Thr Gly Leu Gly Ala Ile Gly Pro Ser
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<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
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atgattgtgc aagtgctgtt taacattgca gataccctgc tgggtggcct gccaactgat 60
ctggtgagca atgctggtgg tcagttgttt tatggcagac cacaagtgag tgaaaatggt 120
gaaccaagtg tgaaactgta tactagtgtg gaagcagcac agctggatca aggtgtgacc 180
attccacatg atattgatct gggtctgagt gcagtgaccc tgcaagattt tgataatcag 240
catctgcaag atagacctac cccaagccct gccccggcga gaccaattac caactggaga 300
agtggtgatg tagtttgggt gaccctgcca agtgcagaat atgcacagag tcagagtgcc 360
atgggcagcc atcctgccta ttggagtgaa gaagccacca ttattaatgt ggccactggt 420
cagagagcaa gtgtgagcag cattaaatgg gatcaagtga ccctgaatgg caaagccctg 480
cataaagaaa cccatagtgg cctggtgtat tatcagctgc cactgatggg caaaattagc 540
ttttggcagc aaggcaccac caaagctggc tatacctata actataacac cactgatagt 600
gatagcctgt gggtttggtg ggatggtgca agcaaagcct atctgtatct gagcacctat 660
accactatgc tgggtgctgg ccctgtcaac attactggcc tgggtgccat tggaccaagc 720
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<213> 人工序列(Artificial Sequence)
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<211> 31
<212> DNA
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Claims (10)
1.一种大鼠HEV-C1 ORF2重组蛋白，其特征在于，其氨基酸序列如SEQ ID No.1所示；
或者其氨基酸序列与SEQ ID No.1所示序列具有至少90％的同一性。
2.编码权利要求1所述的大鼠HEV-C1 ORF2重组蛋白的基因，其特征在于，所述基因的核苷酸序列如SEQ ID No.2所示。
3.一种大鼠HEV-C1 ORF2重组蛋白的表达载体，其特征在于，包括初始载体和权利要求2所述的基因。
4.根据权利要求3所述的大鼠HEV-C1 ORF2重组蛋白的表达载体，其特征在于，所述的基因插入所述初始载体的多克隆位点。
5.一种表达权利要求1所述的大鼠HEV-C1 ORF2重组蛋白的重组菌株，其特征在于，所述重组菌株转化有权利要求3或4所述的表达载体。
6.权利要求1所述的大鼠HEV-C1 ORF2重组蛋白在制备预防人感染大鼠HEV的疫苗中的应用。
7.权利要求1所述的大鼠HEV-C1 ORF2重组蛋白在制备检测人感染大鼠HEV的试剂中的应用。
8.权利要求1所述的大鼠HEV-C1 ORF2重组蛋白免疫动物后获得的单克隆抗体和/或多克隆抗体。
9.权利要求8所述的单克隆抗体和/或多克隆抗体在制备检测大鼠HEV的试剂中的应用。
10.权利要求8所述的单克隆抗体和/或多克隆抗体在制备预防人感染大鼠HEV的疫苗中的应用。
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