人源抗丙型肝炎病毒噬菌体抗体库的构建及筛选

Construction and screening of human hepatitis C virus-specific phage antibody combinatorial library

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。

Aim Human monoclonal antibodies to hepatitis C virus NS4 mosaic antigen were generated from phage antibody combinatorial library. Methods Employing routine RTPCR, the human Fd and κ light chains genes were amplified in human peripheral blood lymphocytes from patients infected with HCV by sets of oligonucleotide primers. Phage antibody library was constructed with the Fd and κ chain genes using pComb3 as vector. The affinity selection and ELISA were adopted for identification of specific phage antibodies to HCV. Results HCVspecific phage antibody library was constructed using Fd and κ genes and pComb3 vector. The library size was about 12×107. The HCVspecific phage antibodies were highly enriched after five rounds of biopanning against HCV NS4 mosaic antigen and positive clones were detected upto 96% by ELISA. Conclusion HCVspecific antibody library and human monoclonal antibodies to HCV can be very useful as molecular tools for research diagnosis and therapy of HCV infection.