丙型肝炎病毒包膜蛋白E2可溶性单链可变区抗体在大肠杆菌中的表达

Expression of Soluble Human ScFv Against Envelope Protein E2 of Hepatitis C Virus Antigen in E.coli.

利用分子生物学技术 ,构建表达丙型肝炎病毒 (HCV)包膜蛋白E2的人源单链可变区抗体 (ScFv)的原核表达载体 ,并在大肠杆菌JM10 9中表达可溶性的HCV E2 ScFv。以重组的HCVE2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 E2 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Ncol/NotI酶切鉴定后 ,该ScFv基因由 75 0bp组成 ,将其亚克隆到pCANTAB5E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HCVE2单链可变区抗体的表达。酶联免疫吸附法 (ELISA)证实表达的HCV E2 ScFv具有与重组HCVE2蛋白的反应活性和特异性 ,对转化的JM10 9大肠杆菌上清中表述的HCV E2 ScFv进行聚丙烯酰胺凝胶电泳 (PAGE) ,证实表达的HCV E2 ScFv的分子量为 2 8kD。为应用HCV E2 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 E2 protein of hepatitis C virus (HCV-E2-ScFv),and express soluble HCV-E2-ScFv in E.coli JM109,using phage display technique,the recombinant phages were panned by recombinant E2 antigen which was coated in a microtiter plate,after five rounds of biopanning,46 clones were identified specific to E2 antigen.750 bp fragment could be released from the plasmid of positive phage colones,and the sequenle analysis indicated that we have got the ScFv BNA fragment.And then DNA fragment was inserted into 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-E2-ScFv is 28kD by SDS-polyacrymide gel electrophoresis(PAGE).The expressed HCV-E2-ScFv will be useful in the immunohistochemical study of liver tissue and gene therapy against HCV infectiion.