|
抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达 |
| |
陈晓穗 朱迎春 王欲晓 刘晓琳 周丽君 王琰
摘 要 目的：尝试将1株来源于人源性噬菌体抗体库的抗HBsAg人Fa b，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ELI S A、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24 h 1 μg/106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2.1×109 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HBsAg人IgG1，为其进一步应用打下了基础。
关键词 噬菌体抗体 乙肝表面抗原 人抗体 真核表达
中国图书分类号 R392-11
Construction and eukaryotic expression of anti -HBsAg human IgG
CHEN Xiao-Sui ZHU Ying-Chun WANG Yu-Xiao
（Navy General Hospital, Beijing100037）
Abstract Objective：To convert anti-HBsAg human F ab derived from phage antibody l ibrary to whole human IgG molecules expressed by eukaryotic cells.Met hods：A syn thesized human Vκ leader sequence was spliced to anti-HBsAg VH and Vκ by over lap PCR.Eukaryotic expression vectors were constructed and transfected into CHO(DHF R)cells.The expressed IgG was analyzed by ELISA,RT-PCR and Western blot. DH F R/MTX system and metal ion induction were used to ampify IgG production.Results：Stable transfectant were obtained after selection and subcloning among which th ree produced human IgG up to 1.0 μg/106/24 h.The expressed human IgG was pr ov ed to bind to HBsAg specifically, contain intact Fc portion and possess an assoc iation constant of 2.1×109 M-1by ELISA, RT-PCR and Weastern bolt an alysi s. The expression level could be increased up to 20 times by DHFR/MTX amplificat ion system and metal ion Zn2+and Cd2+induction. Conclution：These res ults proved the successful conversion of anti-HBsAg human Fab fragments to whol e hIgG molecules with good specificity and affinity.
Key words Phage antibody HBsAg Human antibody Eukaryot ic expression
乙型病毒性肝炎是一种发病率高且危害性大的疾病，抗乙肝病毒表面抗原(H BsAg)的人源 性 抗体在[1] [2] 下一页
上一个医学论文： 小鼠白细胞介素
下一个医学论文： 噬菌体表面呈现抗人红细胞血型A抗原单链抗体的研究
|
|
|
|
|
|
|
您现在的位置： 绿色健康网 >> 医学论文 >> 基础医学论文 >> 正文