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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: CHO cells express anti-human CD80 murine human chimeric antibody ch4E5 and investigate their biological function of blocking CD80-mediated costimulation signaling in vitro. Methods: The expression vector pIRES / ch4E5 containing chimeric heavy and light chain genes was constructed. The expression vector was transfected into 293T cells. After transient expression of ch4E5 antibody was detected by FCM, the expression vector was transfected into CHO cells to construct stable expression cell line CHO ch4E5 antibody was purified from serum-free culture supernatant of CHO-ch4E5 cells by Protein G affinity chromatography. FCM was used to detect the expression of membrane type CD80 by ch4E5 antibody. The mitochondria-derived PBMCs of normal human peripheral blood mononuclear cells To stimulate cells PBLs of normal peripheral blood lymphocytes were used as reaction cells, and the blocking effect of ch4E5 antibody was analyzed by MTT assay. Results: The transient expression of ch4E5 antibody in 293T cells peaked at 48 hours, and the binding rate of the supernatant to L929-B7-1 cells was 96.0%. Ch4E5 antibody was continuously expressed in CHO-ch4E5 cells. The supernatant of ch4E5 antibody was incubated with L929-B7-1 The binding rate of ch4E5 antibody to L929-B7-1, Daudi, Sub-T cells was 94.1%, 25.7% , 23.5%. The ch4E5 antibody could block CD80-mediated costimulation signal transduction and inhibit the proliferation of allogeneic PBLs in vitro. CONCLUSIONS: Anti-human CD80 murine-human chimeric antibody ch4E5 was successfully expressed in CHO cells and in vitro the chimeric antibody has the biological activity of blocking the CD80 -mediated costimulation signal with the parent antibody.