人源抗丙型肝炎病毒噬菌体抗体库的构建、筛选及表达

Construction,Screening and Expression of Hepatitis C Virus Specific Phage Antibodies Combinatorial Library

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。

By using of routine RT-PCR,the gene of human immunoglobulin Fd and κ light chains were amplified from human peripheral lymphocytes of patient infected with HCV by sets of oligonucleotide primers Phage antibody library was constructed with Fd and κ chain genes using pComb3 as vector The library size was about 1 2×10 7,and the affinity selection and ELISA were adopted for generating specific phage antibodies Specific phage antibodies were highly enriched after five rounds of biopanning selection against HCV NS4 mosaic antigen and 96% positive clones were detected by ELISA screening DNA fragment coding for VH and Vκ derived from a positive clone was sequenced and its product was successfully expressed as soluble Fab which was specific for HCV NS4 mosaic antigen HCV specific antibody library and human monoclonal antibodies to HCV may be useful as molecular tools to study the pathogenesis of HCV infection,as well as for the diagnostic and therapeutic purposes