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用RT PCR方法从人外周血单个核细胞总RNA中分别扩增出CTLA4的胞膜外区基因和Igγ1恒定区基因 ,将它们拼接后插入质粒pcDNA3。将重组的HuCTLA4Ig/pcDNA质粒转染CHO细胞 ,经筛选得到了高效、稳定表达HuCTLA4Ig的克隆。培养上清经ProteinA亲和层析柱纯化后 ,所得蛋白在非还原条件下SDS PAGE呈分子量为 11kD的一条带 ;FACS检测显示该纯化蛋白与B7分子有良好的结合能力 ;在体外能抑制MLR的增殖反应。
The CTLA4 extracellular domain gene and Igγ1 constant region gene were amplified respectively from total RNA of human peripheral blood mononuclear cells by RT-PCR and inserted into the plasmid pcDNA3. The recombinant HuCTLA4Ig / pcDNA plasmids were transfected into CHO cells, and clones were obtained with high efficiency and stable expression of HuCTLA4Ig. The purified supernatant was purified by ProteinA affinity chromatography. The obtained protein showed a band of 11kD with SDS-PAGE under non-reducing conditions. FACS analysis showed that the purified protein had good binding ability with B7, and could inhibit MLR Proliferative response.