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目的 :在丙型肝炎病毒 (hepatitisCvirus,HCV)不同变异株中 ,筛选高保守的能涵盖多种MHC限制类型的T细胞表位 ,并构建重组表位抗原的原核表达质粒。方法 :检索文献并结合MHC限制型 ,从中遴选出功能明确的T细胞表位 ;利用Goldkey软件 ,建立HCV全长蛋白序列数据库 ;从中分别截取各个表位所在区段 ,对所选取的表位进行同源性比较 ;从中选取保守性强的部分表位 ,用化学法合成表位基因 ,插入原核表达载体pBVIL1中 ,构建多个融合表达质粒并在E .coli表达。结果 :从文献中获得HCV免疫优势性T细胞表位 18个 ,在由 5 5条全长HCV序列组成的数据库中进行比较 ,获得了大量有关表位保守性的信息 ,并进行了统计分析 ,所构建的 5个表达质粒pBVIL1_T1～T5经鉴定后 ,在大肠杆菌中均获得了高效表达。结论 :本研究有助于深入认识HCVT细胞表位的特性 ,为表位的筛选提供了方法学依据 ,所选取的表位及原核表达的重组蛋白 ,为HCV治疗性疫苗的实验研究提供了候选抗原。
OBJECTIVE: To screen highly conserved T-cell epitopes that can contain many MHC restricted types and to construct prokaryotic expression plasmids for recombinant epitope antigens in different hepatitis C virus (HCV) variant strains. Methods: We searched the literature and combined with MHC restriction type to select the well-defined T cell epitopes. Using Goldkey software, established the HCV full-length protein sequence database; intercepted the region of each epitope, selected the epitope Homology analysis. One of the most conserved epitopes was selected from them, and the epitope gene was synthesized by chemical method and inserted into prokaryotic expression vector pBVIL1 to construct multiple fusion expression plasmids and expressed in E.coli. Results: Eighteen immunodominant T cells were obtained from the literature and compared in a database of fifty-five full-length HCV sequences. A large amount of information on the conservation of epitopes was obtained and statistically analyzed. The constructed 5 expression plasmids pBVIL1_T1 ~ T5 were identified and highly expressed in Escherichia coli. CONCLUSION: This study is helpful to understand the characteristics of HCVT epitopes and provide a methodological basis for epitope screening. The selected epitopes and prokaryotic recombinant proteins provide candidates for the experimental study of HCV therapeutic vaccines antigen.