CTLA4-FasL双功能融合蛋白分子抑制混合淋巴细胞反应及促进淋巴细胞凋亡

目的 构建人CTLA4-FasL融合蛋白真核表达载体，表达CTLA4-FasL融合蛋白，通过体外实验初步研究其生物学特性。方法 通过特异引物分别扩增出CLTA4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，体外表达纯化。Western blot分析CTLA4-FasL融合蛋白的抗原性。体外细胞结合试验研究其结合特异性配体作用。混合淋巴细胞反应研究其抑制免疫应答的效应。结果 测序证实所扩增的PCR产物分别是CLTA4和FasL胞外区的cDNA，其序列与文献报道相符。成功构建了pcDNA3．1-CTLA4-FasL真核表达载体。Western blot分析结果显示，表达获得的蛋白具有CTLA4和FasL的抗原性。体外细胞结合试验显示，CTLA4-FasL融合蛋白可以分别与Jurkat细胞表面的Fas受体和Raji细胞表面的町分子结合。混合淋巴细胞反应结果显示，该融合蛋白可以有效抑制异基因淋巴细胞的刺激作用及诱导淋巴细胞凋亡，并显示了显著的协同效应。结论 成功构建了CTLA4-FasL融合蛋白真核表达载体，体外表达并纯化了CTLA4-FasL融合蛋白，体外实验证实CTLA4-FasL融合蛋白是一个可以有效抑制免疫应答的双功能分子。

Inhibition of mixed lymphocyte reaction and induction of lymphocytes apoptosis by CTLA4-FasL fusion protein

Objective To obtain CTLA4-FasL fusion protein in vitro via a eukaryotic expression vector containing human CTLA4-FasL chimeric gene and to investigate its in vitro immunosuppressive function. Methods The amplified cDNA fragments of extracellular domain of CTLA4 and FasL, respectively, were in frame ligased and subcloned into the eukaryotic expression vector pcDNA3.1(+) to construct the vector pcDNA3.1-CTLA4-FasL, which was transfected into CHO mammalian cell lines with LipofectAMINE reagents. Western blot was performed to detect CTLA4-FasL fusion protein in the conditioned supernatant of CHO transfectants. The potential of CTLA4-FasL fusion protein binding its cognate ligands was analyzed with flow cytometry and its inhibition of lymphocyte response was evaluated through mixed lymphocyte reaction (MLR). Results The amplified fragments were identified with the published sequences. The restriction map confirmed that the vector pcDNA3.1-CTLA4-FasL was correctly constructed. The results of Western blot indicated that CTLA4-FasL fusion protein contains the extracellular domains of both CTLA4 and FasL. CTLA4-FasL fusion protein could bind its cognate ligands which were Fas receptor on the surface of Jurkat cell lines and B7 antigen on the surface of Raji cell lines, respectively. The results of MLR demonstrated that CTLA4-FasL fusion protein was capable of both inhibiting allogeneic MLR at a low concentration and inducing substantial apoptosis of lymphocytes. Conclusion CTLA4-FasL fusion protein, we reported here, is an effectively immunosuppressive immunomodulator, thus worthy of being further investigated its potentials in the induction of transplantation tolerance and the treatment of autoimmune diseases.