丙型肝炎病毒非结构蛋白NS5抗独特型人源单链可变区抗体的筛选与鉴定

Preparation and identification of anti-idiotypic antibody single chain variable fragment against HCV NS5

目的 制备丙型肝炎病毒 (HCV )非结构蛋白NS5 (NS5 )的抗独特型单链可变区抗体scFv(抗 IdscFv) ,为研制HCVNS5的抗 IdscFv疫苗奠定基础。方法 采用噬菌体表面展示技术 ,将HCVNS5单克隆抗体固相包被于Nunc板 ,从噬菌体单链可变区抗体库中经过 5轮“吸附 洗脱 扩增”筛选过程 ,随机挑选 80个克隆 ,利用酶联免疫吸附试验 (ELISA)、交叉反应和竞争抑制实验 ,对其进行免疫学检测 ,获得与HCVNS5单克隆抗体结合活性较强的抗 IdscFv阳性克隆 ,并对HCVNS5特异性抗 IdscFv的编码序列进行序列测定分析。结果 筛选得到的HCVNS5抗 IdscFv片段由 786bp组成 ,具有结合HCVNS5单克隆抗体的生物学活性和特异性。结论 用噬菌体抗体库技术能够成功地获得HCVNS5的抗 IdscFv。本实验结果为开展用抗 IdscFv防治丙型肝炎的研究创造了条件。

Objective To screen anti idiotypic single chain variable fragment (anti Id scFv) against hepatitis C virus non structural protein 5 (HCV NS5) so as to lay the foundation for developing anti Id scFv vaccine of the hepatitis C virus. Methods The semi synthetic phage antibody library was panned by hepatitis C virus non structural protein 5 monoclonal antibody which was coated in a microtiter plate, after five rounds of biopanning, 80 clones specific to HCV NS5 antibody were determined with the enzyme linked immunosorbent assay (ELISA). The specificity of anti idiotypic scFv was identified by ELISA and competitive inhibition assay. The DNA sequence of the positive clone was determined.Results HCV NS5 anti Id scFv has a specific combination character with hepatitis C virus NS5 monoclonal antibody. The DNA sequence data showed that the anti Id scFv encoding gene included 786 bp.Conclusions The anti Id scFv fragments to HCV NS5 monoclonal antibody can be successfully selected by phage display technique, which paves the way for the study of prevention and treatment of hepatitis C using anti Id scFv.