CD137(人4-1BB)在dhfr-CHO中的表达及活性研究

目的 :构建CD137真核高效表达系统 ,深入研究CD137及其配体在细胞信号转导中的作用。方法 :将构建有CD137全长cDNA序列的pCDNA3质粒 (CMV ILA SEN ,CIS)及pSV2 dhfr(含二氢叶酸还原酶基因 ) ,运用脂质体介导法共转染二氢叶酸还原酶缺陷的CHO细胞 (dhfr CHO) ;用G4 18筛选出阳性克隆 ;MTX压力选择系统诱导CD137在dhfr CHO细胞的高效表达 ;RT PCR、免疫细胞化学法及流式细胞术测定CD137的表达情况 ;3H TdR掺入法进行活性研究。结果 :CD137为膜型表达 ,CIS转化dhfr CHO细胞CD137表达率为 96 0 7% ;活性研究显示CD137膜蛋白轻度促进抗CD3单抗诱导的PBMC增殖。结论 :获得高效表达CD137的CHO细胞株 ,CD137膜蛋白促进抗CD3单抗诱导的PBMC增殖。

Expression on dhfr-CHO cells and activity study of CD137 (human 4-1BB)

Objective:To set up a eukaryotic system for high expressing human CD137 and to investigate the role of CD137 and CD137L on signals transduction of cells.Methods:The pCDNA3 plasmid containing full length of human CD137 cDNA sequence(CMV ILA SEN,CIS) and pSV2 dhfr plasmid were cotransfected into dhfr CHO cells by lipoid mediating method. The positive clone was selected with G418. Expression of CD137 on dhfr CHO cells were induced by MTX and detected by RT PCR, immunocytochemistry and flow cytometry.It's activity study was done by method of incorporating 3H TdR.Results:CD137 expressed on the surface of dhfr CHO, expression rate was 96.07%, it's activity study indicated that CD137 increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.Conclusion:dhfr CHO cells that highly express CD137 were established. CD137 can increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.