大鼠CTLA4-FasL融合基因的克隆、表达及体外生物活性研究  

Cloning and expression of rat CTLA4-FasL fusion gene and its biological activity in vitro

作　　者：张瑾[1] 王博[1] 王越[1] 汪习[1] 阎瑾琦[1] 徐元基[1] 于继云[1] 张巍[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《军事医学》2011年第1期58-62,77,共6页Military Medical Sciences

摘　　要：目的表达并纯化CTLA4-FasL重组融合蛋白,鉴定其体外生物活性,为今后将该重组基因用于真核或者病毒载体递送系统的体内研究做准备。方法用RT-PCR方法从大鼠脾细胞中钓取CTLA4和FasL胞外区基因,搭建二者融合的重组基因,以pGEX-6P-1载体表达和纯化融合蛋白;去除GST标签,获得的目的蛋白经体外流式检测分析与细胞表面的相应配体B7和受体Fas分子的结合;CCK8检测系统分析其抑制T淋巴细胞的增殖作用。结果与结论成功调取了目的基因,获得并纯化了CTLA4-FasL目的蛋白,该蛋白既可与细胞表面的B7配体结合,又可与细胞表面的Fas受体结合,并能明显抑制T淋巴细胞的增殖,为今后动物体内研究奠定了基础。Objective To express and purify the recombinant fusion protein and identify its biological activity in vitro.Methods The ectodomain genes of CTLA4 and FasL were cloned from rat spleen cells using RT-PCR and the recombinant fusion gene was constructed and expressed with pGEX-6P-1 vector.The recombinant protein was purified and the GST tag removed to obtain target protein,which was subjected to the analysis of its binding activity to B7 or Fas on the cell surface by FACS and its inhibition of the proliferation of activated T lymphocytes using CCK8 kit.Results and Conclusion The target genes were obtained,and the target CTLA4-FasL proteins were purified that showed not only significant combination with B7 ligand and Fas receptor on the cell surface,but also marked capacity to inhibit the proliferation of T lymphocytes.

关 键 词：CTLA4 FASL 克隆 表达 生物活性 

分 类 号：R392.2[医药卫生—免疫学]