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目的 构建人丙型肝炎病毒 (HCV)特异性噬菌体抗体库 ,制备人源抗HCV单克隆抗体 (mAb)。方法 用常规RT PCR法 ,直接从 5例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 ,与噬菌体载体pComb3连接 ,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。结果 RT PCR可有效地扩增出Fd和κ基因 ,并以此构建成容量为 1 2× 10 7的噬菌体抗体库。经 5轮亲和选择可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 %。结论 抗HCV噬菌体抗体库的构建和人源抗HCVmAb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
Objective To construct human HCV-specific phage antibody library and prepare human anti-HCV monoclonal antibody (mAb). Methods Fd gene and kappa light chain gene were amplified directly from peripheral blood mixed lymphocytes of 5 patients with hepatitis C by conventional RT PCR and linked to the phage vector pComb3 to construct the phage antibody Fab library. Antibody against HCV phage was identified by ELISA after affinity selection of 5 cycles of adsorption-elution-amplification of the antibody library. Results RT PCR can effectively amplify Fd and κ genes and construct a phage antibody library with a capacity of 12 × 10 7. After 5 rounds of affinity selection, the specific phage antibody was highly enriched, and the anti-HCV phage antibody positive clone reached 96%. Conclusion The construction of anti-HCV phage antibody library and the preparation of human anti-HCV mAb provide an effective molecular tool for the diagnosis, treatment and pathogenesis of HCV infection