抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达

Construction and Eukaryotic Expression or Anti-HBsAg Human IgG

目的:尝试将一株来源于人源性噬菌体抗体库的抗HBsAg人Fab,转换成完整的抗HBsAg人IgG.方法:采用重叠PCR方法,在抗HBsAg人Fab的V_κ和VH基因的5’端接上人工合成的人_κ链的前导序列,构建表达完整IgGl的真核表达载体,转染CHO(DHFR^-)细胞,用ELISA、RT-PCR和免疫印迹检测抗HBsAg人IgG的表达.通过DHFR/MTX体系及金属离子诱导提高Ig表达.结果:获得表达量在lμg/10~6细胞/24小时的稳定表达抗HBsAg人IgG的转染细胞系.并证实所表达人IgG具有良好特异性及完整Fe段,其亲和力约为2.1×10~9 M^(-1).又通过DHFR/MTX扩增系统的作用及金属离子锌和镉对小鼠金属硫蛋白启动子的激活作用,使IgG的表达量提高了约20倍.结论:通过拼接人κ链的前导序列在真核表达系统成功地将抗HBsAg人Fab转换成完整的抗HBsAg人IgGl,为其进一步应用打下了基础.

Objectives To convert anti-HBsAg human Fab derived from phage antibody library to whole human IgG molecules expressed by eukaryotic cells. Methods: A synthesized human Vk leader sequence was spliced to anti-HBsAg VH and Vk by overlap PCR. Eukaryotic expression vectors were constructed and transfected into CHO(DHFR-) cells. The expressed IgG was analyzed by ELISA, RT-PCR and Western blot. DHFR/MTX system and metal ion induction were used to amplify IgG production. Results: Stable transfectant were obtained after selection and subcloning among which three produced human IgG up to 1. 0μ g/106/24 hrs. The expressed human IgG was proved to bind to HBsAg specifically, contain intact Fc portion and possess an association constant of 2.1×109M-1 by ELISA, PT-PCR and Weastern blot analysis. The expression level could be increased up to 20 times by DHFR/MTX amplification system and metal ion Zn2+ and Cd2+ induction. Conclution: These results proved the successful conversion of anti-HBsAg human Fab fragments to whole IgG molecules with good specificity and affinity.