摘要
利用分子生物学技术 ,构建表达丙型肝炎病毒 (HCV)核心蛋白的人源单链可变区抗体 (ScFv)的原核表达载体 ,并在大肠杆菌JM 10 9中表达可溶性的HCV core ScFv。以重组的HCV核心蛋白为包被抗原 ,利用噬菌体抗体库的表面展示技术 ,筛选到含有HCV core ScFv基因的噬菌体克隆。从噬菌体抗体阳性克隆中提取质粒 ,经NcoI/NotI酶切鉴定 ,该ScFv基因由 75 0bp组成。将其亚克隆到 pCANTAB5E表达载体中 ,转化大肠杆菌JM10 9,提取质粒进行DNA序列测定 ,符合ScFv的重链可变区和轻链可变区基因结构特点。IPTG诱导转化的大肠杆菌JM10 9,在其培养上清中获得了可溶性HCV core ScFv的表达。酶联免疫吸附法 (ELISA)证实表达的HCV core ScFv具有重组HCV核心蛋白的反应活性和特异性。对转化的JM10 9大肠杆菌上清中表达的HCV core ScFv进行聚丙烯酰胺凝胶电泳 (PAGE) ,证实表达的HCV core ScFv的分子量为 2 8kDa。为应用HCV core ScFv进行肝组织免疫组织化学和细胞内免疫基因治疗研究奠定了基础。
To construct expressive vector for human ScFv against core protein of hepatitis C virus (HCV core ScFv),and to express soluble HCV core ScFv in E.coli JM109. Using phage display technique, the recombinant phages were panned by recombinant core antigen which was coated in a microtiter plate, and after five rounds of biopanning, 86 clones were identified specific to core antigen. 750 bp fragment could be released from the plasmid of positive phage clones, and the sequence analysis indicated that we have obrained the ScFv DNA fragment. Then DNA fragment was inserted into the expressive vector pCANTAB5E, and E. coli host JM109 was transformed and induced by IPTG. The specificity of ScFv in the culture medium was evaluated by enzyme linked immunosorbent assay (ELISA).The molecular weight of expressed HCV core ScFv protein is 28 000 dalton as determined with the aid of SDS polyacrymide gel electrophoresis (PAGE). The expressed HCV core ScFv protein will be useful in the immunohistochemical study of liver tissue from patients with hepatitis C and gene therapy against HCV infection.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2000年第6期394-397,共4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39900130)
