HCV NS3蛋白单克隆抗体的制备及其识别区域的分析

目的 制备针对丙型肝炎病毒（HCV）非结构区NS3全长蛋白的单克隆抗体（MAb），并分析获得的单抗识别表位所在区域，为建立以NS3蛋白为靶位的抗HCV研究提供抗体工具。方法 用原核表达的HCV非结构区NS3全长蛋白作为免疫原，采用小鼠腹股沟皮下NC膜包埋法免疫小鼠，按常规杂交瘤细胞的制备方法，经细胞融合、克隆化制备抗NS3蛋白的MAb。用间接免疫荧光法和Westem blot鉴定其特异性。分别构建NS3丝氨酸蛋白酶（NS3蛋白的N末端1／3）编码基因的真核表达质粒pcDNA3．1（-）-ns3p、NS3解旋酶（NS3蛋白的C末端2/3）编码基因的真核表达质粒pcDNA3．1（-）-ns3A，将其瞬时转染COS-7细胞后，以获得的单克隆抗体作为一抗，通过免疫荧光分析获得单抗识别表位所在的区域。结果 获得了2株抗NS3蛋白的单克隆抗体，这2株MAbs均特异识别NS3蛋白，并确定了它们的结合区域。结论 获得了针对NS3蛋白的单克隆抗体，并对其单抗识别表位所在的区域进行了分析，为下一步进行以NS3蛋白为靶位的抗HCV研究奠定了良好的基础。

Preparation of monoclonal antibody against HCV NS3 protein and determination of its target region

Objective To prepare the monoclonal antibody (MAb) against HCV full NS3 protein and detect their binding regions, for anti-HCV research based on NS3 protein. Methods Balb/c mice were immunized with the recombinant NS3 protein. The specific monoclonal antibodies (MAbs) were prepared by the cell fusion technique. The specificity of the MAbs was identified by immunofluorescent assay (IFA) and Western blotting. Indirect ELISA was used to detect the titer and Ig subclass of the MAbs. The eukaryotic expression vectors pcDNA3.1(-)-ns3t_ p and pcDNA3.1(-)-ns3t_ h respectively containing serine protease and helicase gene were constructed. COS-7 cells were transiently transfected by the two plasmids by lipofectamine method, and then the binding regions of the two MAbs were determined by IFA. Results Two strains of hybridoma secreting the anti-NS3 protein MAbs were prepared. The MAbs could specifically bind to different regions of the NS3 protein. Conclusion The specific MAbs lay a foundation for carrying out the anti-HCV research based on NS3 protein.