HCV NS3诱导小鼠特异性T细胞反应的抗原多肽序列筛选

目的 筛选HCV NS3的小鼠T细胞表位多肽序列.方法 BALB/c小鼠用HCV NS3加po-ly （I∶C）或CpG ODN及Montanide ISA720佐剂免疫后,分离其脾淋巴细胞,以覆盖HCV NS3全基因的合成重叠多肽组成的不同多肽库进行刺激,以ELISPOT及流式细胞术检测分泌干扰素γ（IFN-γ）的细胞数及CD8＋/IFN-γ＋细胞或CD4 ＋/IFN-γ＋细胞百分比,确定抗原性最强的多肽库,找出HCV NS3的小鼠T细胞表位多肽.结果 通过ELISPOT及流式细胞术检测,能够刺激淋巴细胞分泌IFN-γ最多及CD8 ＋/IFN-γ＋细胞或CD4＋/IFN-γ＋细胞百分比最高的多肽被选出作为阳性多肽,其中的一个多肽经进一步的ELISPOT及流式细胞术检测确认,其序列为GGCSGGAYDⅢCDECHS.结论 HCV NS3小鼠T细胞表位序列的确定为HCV疫苗的研究打下了基础.

Epitope screening of HCV NS3 inducing antigen-specific T cell response in mice

Objective To select murine T cell epitope of HCV NS3.Methods Femalebalb/c mice were vaccinated by recombinant HCV NS3 protein adjuvanted by poly（Ⅰ∶C）and Montanide ISA 720 or CpG ODN and Montanide ISA 720.Splenocytes from vaccinated mice were stimulated by a variety of peptide pools composed of synthetic overlapped peptides covering the whole gene of HCV NS3.ELISPOT and flow cytometry were applied to identify the peptide pools that produced most abundant IFN-γ spot forming units and/or CD8 ＋/IFN-γ ＋ or CD4 ＋ /IFN-γ＋ cell ratios.Results HCV NS3 peptide pools that produced the most abundant IFN-γ spot forming units were selected by ELISPOT and the highest splenocyte ratio of CD8 ＋/IFN-γ ＋ or CD4 ＋/IFN-γ＋ cell by flow cytometry.The epitope sequences were retrieved in the peptide pool composition matrix.One peptide sequenced as GGCSGGAYDⅢCDECHS was selected as one of the mice T cell epitopes and further confirmed by ELISPOT assay and flow cytometry.Conclusion The identification of HCV NS3 mice T cell epitope facilitates the subsequent study for HCV vaccine development.