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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。
The prokaryotic expression vector of human single chain variable region antibody (ScFv) expressing hepatitis C virus (HCV) envelope protein E2 was constructed by using molecular biology technique and the soluble HCV E2 ScFv was expressed in Escherichia coli JM109. Using recombinant HCVE2 as coating antigen, phage clones containing HCV E2 ScFv gene were screened by surface display technology of phage antibody library. Plasmids were extracted from phage antibody positive clones and identified by Ncol / NotI digestion. The ScFv The gene consisted of 75 0bp and was subcloned into pCANTAB5E vector and transformed into E. coli JM109. The plasmid was extracted for DNA sequencing, which was in accordance with the gene structure of ScFv. IPTG-induced transformation of E. coli JM109, the expression of soluble HCVE2 single chain variable region antibody was obtained in the culture supernatant. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the expressed HCV E2 ScFv has reactivity and specificity with the recombinant HCVE2 protein, and polyacrylamide gel electrophoresis (PAGE) of HCV E2 ScFv expressed in the transformed JM109 E. coli supernatant ), Demonstrating that the expressed HCV E2 ScFv has a molecular weight of 28 kD. It laid the foundation for the study of liver tissue immunohistochemistry and intracellular immunotherapy with HCV E2 ScFv.