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