抗角蛋白Fab抗体在大肠杆菌中的表达与复性研究

Expression of human Fab antibody against keratin in E.coli and its renaturation

目的 :用大肠杆菌分别表达抗角蛋白抗体的轻链和Fd片段 ,体外复性得到Fab抗体。方法 :从已构建的质粒p3MH/Fab中 ,亚克隆抗角蛋白抗体的轻链和Fd片段基因 ,并分别插入载体pET32a中 ,构建重组质粒。以重组质粒分别转化大肠杆菌BL2 1(DE3) ,在IPTG诱导下进行表达。SDS PAGE分析发现 ,在相对分子量 (Mr)为 2 5 0 0 0处有外源蛋白表达。轻链和Fd片段变性后等量混合于折叠液中 ,复性后形成Mr 约为4 5 0 0 0的蛋白。结果 :成功地表达抗角蛋白抗体的轻链和Fd片段 ,ELISA和Westernblot证实 ,复性产物具有与角蛋白结合的能力。结论 :获得了具有活性的抗角蛋白的Fab抗体 ,为其应用研究打下了基础 ,也表明包涵体表达基因工程抗体技术是可行的。

AIM: To express Fd fragment and L chain of human anti-keratin Fab in E.coli BL21(DE3) respectively and obtain human anti-keratin Fab by renaturation in vitro. METHODS: Genes of L chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/ Fab were subcloned into vector pET32a respectively. The recombinant plasmids pETL and pETFd were transformed into E.coli BL21(DE3) and induced to express with IPTG. Fd fragment and L chain inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. The renatured Fab was characterized by SDS-PAGE, Western blot and ELISA. RESULTS: Fd fragment and L chain of human anti-keratin Fab were efficiently expressed. ELISA and Western blot showed that the renatured Fab could bind with human keratin. CONCLUSION: The successfully prepared anti-keratin Fab with binding activity to human keratin laid a solid foundation for its further application.