摘要
目的 对模拟人卵巢癌抗原的鼠源性抗独特型单链抗体6B11scFv进行人源化改造和表达。方法采用重叠PCR和基因工程技术,将6811scFv基因与人IgG1铰链区和CH3区的基因进行融合,构建人源化抗独特型抗体6811VHVLhC基因。用IPTG诱导大肠杆菌进行表达,并采用酶切法、DNA测序、SDS—PAGE电泳、ELISA和免疫印迹技术等进行鉴定。结果酶切和DNA测序表明插入片段正确;SDS—PAGE凝胶电泳显示融合蛋白分子量50 000 Da处有一诱导蛋白带;而不连续非变性凝胶电泳表达产物分子量为100 000 Da;ELISA和免疫印迹结果显示,表达出的6811VHVLhC人源化抗体仍具有6B11scFv和人IgG1CH3的活性。结论 已成功构建并表达出双价的可模拟卵巢癌抗原的人源化抗独特型抗体。
Objective To humanize and express an anti - idiotypical single chain antibody (6BllscFv) , which mimicing ovarian cancer antigen. Methods The gene of humanized anti - idiotypical antibody 6B11VHVLhC was constructed in prokaryolic expression vector by genetic fusion of 6BllscFv to human IgGl hinge and CHS region, using overlap PCR and DNA recombinant technique. The fusion protein was induced with IPTG in E.coli and analyzed with enzyme digestion, DNA sequencing, SDS -PAGE, ELISA and western blot. Results The results of enzyme digestion and DNA sequencing showed that the fusion gene was correctly inserted and the expressed protein with molecular weight of 50 000 Da could be seen as the expected size in SDS - PAGE gel, while 100.000 Da in non- denaturing system. The fusion protein still remained immune activities of both 6B11scFv and human CH3 of IgG1 by analysis of ELISA and Western blotting. Conclusions The anti - idiotypical single chain antibody 6B11scFv has successfully be humanized as a bivalent minibody and expressed with activities of both 6B11 and human IgG1.
出处
《中国妇产科临床杂志》
2002年第1期44-47,共4页
Chinese Journal of Clinical Obstetrics and Gynecology
基金
国家863基金资助(项目编号102-09-02-07)
关键词
抗独特型抗体
人源化
人IgG1
卵巢肿瘤
融合蛋白
Anti- idiotypic antibody
Ovarian neoplasms
Humanized
Human IgG1
Reeombinant fusion protein
