CTLA-4-FasL融合分子的构建及其生物学特性的鉴定

文章通过特异的引物分别扩增出CTLA 4和FasL胞外区的cDNA ,将它们拼接后 ,克隆入真核表达载体pcDNA3 1( + )中 ,进行表达、纯化 ,获得CTLA 4 FasL融合蛋白。Westernblot分析显示了该融合蛋白具有CTLA4 胞外区和FasL胞外区的抗原性。体外试验表明 ,该融合蛋白可以结合Jurkat细胞表面的Fas受体和Raji细胞表面的B7分子 ,表明了该分子双特异性的特点。该融合蛋白能够直接诱导Jurkat细胞发生凋亡 ,且此凋亡效应伴随Raji细胞的参与而增强 ,初步证实了该分子的免疫抑制效应 ,从而为进一步研究该融合蛋白特性及应用奠定了基础。

Construction and Characterization of Human CTLA-4-FasL Fusion Protein

The amplified cDNA fragments of extracellular domain of CTLA 4 and FasL were in frame ligased together and were subcloned into the eukaryotic expression vector pcDNA3 1(+) to construct the plasmid phCFL,which was transfected into CHO mammalian cell lines with Lipofectin Western blot was performed to detect the CTLA 4 FasL fusion protein in the conditioned supernatant of CHO transfectants CTLA 4 FasL fusion protein could bind its cognate ligands which are Fas receptor on the surface of Jurkat cell lines and B7 antigen on the surface of Raji cell lines,respectively Furthermore,CTLA 4 FasL fusion protein induced apoptosis of Jurkat cell lines after engaged with Fas antigen,and its potential was enhanced after addition of Raji cell lines to the reaction system In conclusion,CTLA 4 FasL fusion protein,we reported here,is an effective negative immunomodulator,thus worthy of being further investigated its potentials in the induction of transplantation tolerance and treatment of autoimmune diseases