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目的制备针对HCV NS3解旋酶的单克隆抗体,分析其识别抗原表位及潜在生物学功能,为HCV诊断和治疗提供关键材料。方法制备截短型重组HCV NS3解旋酶(rNS3,aa1192-1459,1b型),免疫小鼠,按常规方法制备单克隆抗体。采用ELISA和Weston Blot筛选抗rNS3的单克隆抗体。采用29条重叠7个氨基酸的16肽和9条短肽,通过间接ELISA和竞争ELISA测定单抗识别表位。采用HCV(JFH1克隆,2a型)感染huh7.5.1细胞免疫荧光染色法,鉴定单克隆抗体对天然抗原构象的识别特性。结果以rNS3筛选,共获得29株ELISA阳性单克隆抗体,其中27株Weston Blot阳性。
OBJECTIVE: To prepare a monoclonal antibody against HCV NS3 helicase and to identify its epitopes and potential biological functions for the diagnosis and treatment of HCV. Methods A truncated recombinant HCV NS3 helicase (rNS3, aa1192-1459, lb) was prepared and immunized to prepare the monoclonal antibody according to the routine method. Anti-rNS3 monoclonal antibodies were screened by ELISA and Weston Blot. Monoclonal anti-epitopes were determined by indirect ELISA and competition ELISA using 29 overlapping 16 amino acids and 9 short peptides. Huh7.5.1 cells were infected with HCV (JFH1 clone, type 2a) by immunofluorescence staining to identify the recognition characteristics of the monoclonal antibody against the native antigen conformation. The results of rNS3 screening, a total of 29 ELISA positive monoclonal antibodies, of which 27 Weston Blot positive.