摘要
为探索新型治疗方法 ,制备抗丙型肝炎病毒(HCV)非结构蛋白NS4A的抗独特型人源单链可变区抗体 (抗 IdscFv) ,采用噬菌体表面展示技术 ,将抗HCVNS4A单克隆抗体固相包被于Nunc板 ,应用半合成的人源单链可变区抗体文库技术 ,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 82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HCVNS4A单克隆抗体结合活性较强的抗 IdscFv阳性克隆 ,并对HCVNS4A特异性抗 IdscFv的编码基因进行序列测定分析。结果 ,经过筛选 82个克隆中有 4 0株克隆ELISA的吸光度 (A4 5 0nm)值较高 ,将这些噬菌体上清与牛血清白蛋白 (BSA)进行交叉反应 ,确定其中有 7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为 789bp。本实验结果提示 ,用噬菌体抗体库技术能够成功地获得抗HCVNS4A的抗 IdscFv,为开展用抗
To screen anti idiotypic single chain variable fragments(anti Id scFv)against hepatitis C virus NS4A(HCV NS4A)so as to lay a foundation for developing anti Id scFv vaccine againist the hepatitis C virus. The recombinant phage antibody library was panned by hepatitis C virus NS4A monoclonal antibody which was coated in a microwell plate. After five rounds of biopanning, 82 clones specific to HCV NS4A antibody were determined with the enzyme linked immunoadsorbent 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. The result showed that HCV NS4A anti Id scFv had a specific combination character with hepatitis C virus NS4A monoclonal antibody. The DNA sequence data showed that the anti Id scFv coding gene included 789 bp. The results suggested that the anti Id scFv fragments to HCV NS4A monoclonal antibody could be successfully selected by recombinant phage antibody library screening technique, which might pave a way for the study of new preventive and therapeutic strategy of hepatitis C using anti Id scFv.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2003年第1期37-39,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金资助课题 (编号C3990 0 1 30 )
