摘要
尝试将 1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Vκ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IgG1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 :获得表达量在每 2 4h 1μg 10 6 细胞的稳定表达抗HBsAg人IgG的转染细胞系。并证实所表达IgG具有良好特异性及完整Fc段 ,其亲和力约为 2 .1× 10 9M 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 2 0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HBsAg人IgG1,为其进一步应用打下了基础。
Objective:To convert anti-HBsAg human Fab derived from phage antibody library to whole human IgG molecules expressed by eukaryotic cells.Methods: A synthesized human Vκ leader sequence was spliced to anti-HBsAg VH and Vκ 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 ampify IgG production.Results: Stable transfectant were obtained after selection and subcloning among which three produced human IgG up to 1.0 μg/10 6/24 h.The expressed human IgG was proved to bind to HBsAg specifically, contain intact Fc portion and possess an association constant of 2.1×109 M -1 by ELISA, RT-PCR and Weastern bolt analysis. The expression level could be increased up to 20 times by DHFR/MTX amplification system and metal ion Zn 2+ and Cd 2+ induction. Conclution:These results proved the successful conversion of anti-HBsAg human Fab fragments to whole hIgG molecules with good specificity and affinity.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2000年第5期248-251,共4页
Chinese Journal of Immunology
基金
国家 8 6 3计划资助项目! (Z 18 0 1 0 2 0 3)
