CD80鼠-人嵌合抗体的CHO细胞表达及体外生物学功能的初步研究

Expression of human-mouse chimeric antibody against CD80 in CHO cells and preliminary study on the biological activity in vitro

目的:CHO细胞表达抗人CD80鼠-人嵌合抗体ch4E5,并研究其在体外阻断CD80介导的共刺激信号转导的生物学功能。方法:构建含有嵌合重、轻链基因的表达载体pIRES/ch4E5;表达载体先转染293T细胞,FCM检测到ch4E5抗体的瞬时表达后,表达载体再转染CHO细胞,构建稳定表达细胞株CHO-ch4E5;ProteinG亲和层析法从CHO-ch4E5细胞无血清培养上清中纯化ch4E5抗体,FCM检测ch4E5抗体对膜型CD80的识别;以丝裂霉素处理的正常人外周血单核细胞PBMCs为刺激细胞,以异体正常人外周血淋巴细胞PBLs为反应细胞,用MTT法分析ch4E5抗体的阻断作用。结果:ch4E5抗体在293T细胞的瞬时表达于48小时达到高峰,上清与L929-B7-1细胞结合率为96.0%;CHO-ch4E5细胞持续表达ch4E5抗体,其培养上清与L929-B7-1细胞结合率为95.7%;3天培养上清中,ch4E5抗体的产率约为5.56mg/L;ch4E5抗体和L929-B7-1、Daudi、Sub-T细胞结合率分别为94.1%、25.7%、23.5%;ch4E5抗体可以阻断CD80介导的共刺激信号转导,抑制异体PBLs的体外增殖。结论:在CHO细胞中成功表达了抗人CD80鼠-人嵌合抗体ch4E5,在体外该嵌合抗体具有亲本抗体阻断CD80介导的共刺激信号的生物学活性。

Objective：To express human-mouse chimeric antibody against human CD80 （ch4E5 antibody） in CHO cells,and to research the activity of ch4E5 antibody for blocking CD80 of the costimldatory signals in vitro. Methods：pIRES/ch4E5 with chimeric heavy and light gene was constructed. Transient expression of ch4E5 antibody in 293 T cells was detected by FCM. CHO cells were transfected by pIRES/ch4E5 to construct stable cell line secreting chimeric antibody. The ch4E5 antibody was purified from culture supematant without fetal calf serum （FCS） by Protein G affinity chromatography, and then binding of ch4E5 antibody to membrane CD80 was detected by FCM. The blocking effect of ch4E5 antibody was determined by MTT assay in which PBMCs pretreated with mitomycinC as the stimulator ceils and ailogeneic PBLs as the respondor cells. Results：Transient expression of ch4E5 antibody,whose combination rate was 96.0% by FCM, peaked at 48 hours in 293T cells. The ch4E5 antibody was stably expressed in CHO-ch4E5 ceils, whose combination rate was 95.7% by FCM. About 5.56 milligram ch4E5 antibodies were purified from one liter culture supematant without FCS. L929-B7-1, Daudi and Sub-T cells were specially identified by ch4E5 antibody, whose combination rates were 94.1%, 25.7 % and 23.5 % respectively by FCM. The costimulatory signals of CD80 could be blocked by ch4E5 antibody, which effectively inhibited proliferation of allogeneic PBLs in vitro. Conclusion： Human-mouse chimeric antibody against human CD80 （ch4E5 antibody） could be secreted from the recombinant CHO cells, and has obvious biological function to block the costimulatory signals of CD80 in vitro as similar as original 4E5 antibody.