摘要
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Mr27000(L)和Mr25000(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50000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。
Objective To investigate the high-level expression and folding of human Fab antibody against hepatitis B virus surface antigen （HBsAg） in E. ooli. 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 M,25 000 and a M,27 000 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 Mr50000 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.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第1期38-39,45,共3页
Immunological Journal
基金
北京市自然科学基金资助项目(7002033)
