丙型肝炎病毒核心蛋白抗独特型人源单链可变区抗体的筛选与鉴定

SCREENING AND IDENTIFICATION OF ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENT AGAINST HCV CORE PROTEIN

为制备抗丙型肝炎病毒核心蛋白的抗独特型单链可变区抗体 (抗 IdscFv) ,采用噬菌体表面展示技术 ,将抗 HCV核心蛋白的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 ,获得可与HCV核心蛋白单克隆抗体结合的抗独特型人源单链可变区抗体的噬菌体集落。随机挑选 6 0个克隆 ,利用酶联免疫吸附法 (ELISA)、交叉反应和竞争抑制实验 ,对其进行免疫学检测和鉴定。获得与HCV核心蛋白单克隆抗体结合活性较强的抗 IdscFv阳性克隆 ,并对HCV核心蛋白特异性抗 IdscFv的编码序列进行测定分析。结果经过 5轮“吸附 洗脱 扩增”筛选 ,在随机挑选的 6 0个克隆中 ,有 2 0株克隆ELISA的吸光度(A4 5 0nm)值较高 ,这些噬菌体上清与牛血清白蛋白 (BSA)进行交叉反应后 ,确定了其中有 6株交叉反应较弱 ,结合 2次ELISA重复实验的A值及竞争抑制实验结果 ,最后确定 1株阳性克隆 ,提取质粒 ,进行DNA序列测定 ,DNA大小为 76 8bp。本实验结果提示用噬菌体抗体库技术能够成功地获得抗 HCV核心蛋白的抗 IdscFv ,为开展用抗

To screen anti idiotypic single chain variable fragments(anti Id scFv)against hepatitis C virus core protein(HCV core)so as to lay a foundation for developing anti Id scFv vaccine against hepatitis C virus. The recombinant phage antibody library was panned by solidified hepatitis C virus core protein specific monoclonal antibody which was coated in a microwell plate. After five rounds of biopanning,60 clones specific to HCV core antibody were determined with the enzyme linked immunoadsorbent assay(ELISA). The specificity of anti idiotypic scFv was identified by ELISA and competition inhibition assay. The DNA sequence of the positive clone was determined. The result showed that HCV core anti Id scFv had a specific combination character with hepatitis C virus core monoclonal antibody. The DNA sequence data showed that the anti Id scFv coding gene included 768 bp. The results suggested that the anti Id scFv fragment to HCV core monoclonal antibody could be successfully selected by recombinant phage antibody technique,which paved a way for the study of a new preventive and therapeutic strategy of hepatitis C by using anti Id scFv.