摘要
目的:制备靶向人表皮生长因子受体3(HER3)的全人源Fab型抗体,并对其亲和力及生物学活性进行鉴定。方法:用Bsp QⅠ限制性内切酶分别将靶向HER3的全人源抗体的完整轻链、重链的可变区全长和CH1恒定区插入载体p3457,构建重组质粒;通过将轻、重链质粒瞬时共转染293E悬浮细胞进行表达,并利用镍柱纯化得到Fab型抗体蛋白;采用ELISA鉴定其与HER3胞外区是否结合,Forte Bio实验鉴定其亲和力,CCK8检测其对MDA-MB-453细胞增殖能力的影响。结果:分别获得表达轻链全长、重链可变区全长及CH1恒定区的HER3抗体重组质粒,并通过瞬时共转染293E悬浮细胞获得分泌型HER3 Fab抗体;ELISA实验证明其可与HER3胞外区蛋白直接结合;Forte Bio实验证明Fab型抗体与人源HER3胞外区蛋白具有一定的亲和力(KD=1.08×10-8mol/L);体外增殖实验表明,该抗体可显著抑制MDA-MB-453体外增殖。结论:通过293E悬浮细胞表达、镍柱亲和纯化,获得具有较高亲和力和功能活性的HER3 Fab型抗体,为HER3高表达型癌症的诊断、治疗提供物质基础。
Objective： To construct and express the anti-human epidermal growth factor receptor 3（HER3） hu- man Fab antibody and evaluate the antibody＇s affinity and biological activities. Methods： Construct recombinant vector p3457-HC which contained the variable and CH1 domain of heavy chain and recombinant vector p3457- LC which contained the whole light chain by using the restriction enzyme BspQ Ⅰ.The HER3 Fab antibody was obtained by eo-transfected 293E cells with p3457-HC and p3457-LC and purifited using nickel column purifica- tion. ELISA was used to identify whethere it can combinded with HER3 extracelluar membrane domain（ECD）. Its affinity was determinded by ForteBio. The ability of HER3 Fab of inhibity the MDA-MB-453 cells growth was evaluated using CCK-8 assay. Results： The recombinant vector p3457-HC and p3457-LC were successfully con- structed and expressed by sequencing and Western blot. ELISA showed that HER3 Fah can combined with HER3 ECD. ForteBio showed that the affinity of HER3 Fab and HER3 ECD was 1.08×10^-8 mol/L. In addition, the HER3 Fah can inhibit MDA-MB-453 cell growth. Conclusion： We successfully constructed and expressd the func- tional HER3 Fah antibody, which lay establishment to the diagnosis and treatment of HER3-upregulated cancer.
出处
《生物技术通讯》
CAS
2015年第5期595-599,共5页
Letters in Biotechnology
基金
国家"十三五"重大专项定向委托课题(2015ZX09501008)
