人源抗HBs基因工程抗体在E. coli中的高效表达与折叠研究

目的 高效表达是基因工程抗体走向临床的关键问题之一.本试验旨在研究人源抗HBs基因工程抗体在E. coli中的高效表达与折叠.方法 本实验将抗HBsAg抗体的轻链(L)和重链Fd段基因,分别克隆于pET20b质粒中并分别转化到大肠肝菌BL21(DE3),IPTG诱导后,SDS-PAGE分析发现在Mr27 000(L)和Mr25 000(Fd)处有外源蛋白表达,表达蛋白含量分别为53%和48%.L链和Fd 包涵体蛋白经盐酸胍变性后,等量混合于折叠液中.结果 L和Fd可复性形成了Mr约50 000的蛋白,ELISA结果表明,复性蛋白具有与HBsAg结合的能力.结论 抗HBsAg抗体Fab段在大肠杆菌中的表达与复性的成功,表明包涵体表达基因工程抗体在技术是可行的.

High-level expression and folding of human Fab antibody against HBsAg in E. coli

Objective To investigate the high-level expression and folding of human Fab antibody against hepatitis B virus surface antigen (HBsAg) in E. coli. Methods The genes of Fd fragment and L chain of human antibody against HBsAg were cloned into plasmid pET-20b. The resultant plasmids of pETFd and pETL were transformed into E. coli BL21(DE3) respectively, resulting in production of a Mr25 000 and a Mr27 000 proteins after induction with IPTG. The expression levels of Fd and L protein were 48% and 53%,respectively. Fd-fragment and L chain inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. Results A Mr50 000 protein was observed by SDS-PAGE and its antigenicity was confirmed by Western blotting. Furthermore, the binding activity of the protein to HBsAg was revealed by ELISA. Conclusion These results indicate that the inclusion strategy of producing Fab fragment is available in technology.