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用常规RT PCR法 ,直接从 5名丙型肝炎患者外周血混合淋巴细胞中扩增抗体重链Fd基因和κ轻链基因 ,构建噬菌体抗体Fab库。对抗体库进行 5轮吸附 洗脱 扩增的亲和选择后 ,以ELISA法鉴定抗HCV噬菌体抗体。 5轮亲和选择使特异性噬菌体抗体得到高度富集 ,抗HCV噬菌体抗体阳性克隆达 96 %。对一阳性克隆在大肠杆菌中表达 ,经ELISA及Westernblot分析鉴定 ,证实成功表达出人源可溶性Fab。对抗体基因VH和VκDNA序列进行测定 ,证实所获基因为抗体可变区基因。抗HCV噬菌体抗体库的构建和人源抗HCV单克隆抗体Fab段的制备 ,为HCV感染的诊断、治疗和发病机制的研究提供有效的分子工具。
The antibody heavy chain Fd gene and kappa light chain gene were amplified directly from peripheral blood mixed lymphocytes of 5 hepatitis C patients by using the conventional RT PCR method to construct the phage antibody Fab library. Antibody to anti-HCV phage was identified by ELISA after the affinity library was subjected to 5 cycles of adsorption and elution amplification. Five rounds of pro- and selection resulted in highly enriched specific phage antibodies, with 96% positive for anti-HCV phage antibody. A positive clone was expressed in E. coli and identified by ELISA and Western blot analysis. The results showed that human-soluble Fab was successfully expressed. Antibody gene VH and V kappa DNA sequences were determined, confirmed that the gene obtained is an antibody variable region gene. The construction of anti-HCV phage antibody library and the preparation of human anti-HCV monoclonal antibody Fab fragment provide an effective molecular tool for the diagnosis, treatment and pathogenesis of HCV infection.