重组FAS分子的表达、纯化及抗体制备

用ＰＣＲ法扩增出编码人ＦＡＳ分子胞外区的ｃＤＮＡ片段，直接克隆到ｐＧＥＭ－Ｔ载体上，经ＤＮＡ序列测定后，再插入到谷胱甘肽转硫酶（ＧＳＴ）融合蛋白表达载体ｐＧＥＸ－ＫＧ的ＥｃｏＲⅠ和ＳａｌⅠ位点之间，构成重组质粒ｐＫＧ－ｈＦＡＳ，将此质粒导入大肠杆菌，经ＩＰＴＧ诱导后获得ＧＳＴ－ｈＦＡＳ重组融合蛋白的表达，用谷胱甘肽偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ经亲合层析获得纯化的ＧＳＴ－ｈＦＡＳ蛋白，经凝血酶酶切和二次亲合层析去除ＧＳＴ部分，得到纯化的ＦＡＳ蛋白．用纯化的ＦＡＳ抗原免疫家兔制备了抗ＦＡＳ抗体，经检测发现抗ＦＡＳ抗体能诱导Ｕ９３７细胞发生细胞凋亡

Expression and Purification of Recombinant FAS Antigen and the Production of Anti FAS Antibodies

FAS is a typeⅠ integral membrane protein belonging to the tumor necrosis factor(TNF) receptor family and tranduces a cell death signal.The aim of this study was to prepare recombinant FAS antigen and its antibodies for the research of its functions in the future.In this work,the DNA fragments of extracellular domain of human FAS molecule were amplified by PCR method,and then cloned into PGEM T vector.After DNA sequencing,it was cloned into GST fusion protein expressive vector PGEX KG,then recombinant plasmid pKG hFAS was constructed and introduced into E.coli .GST hFAS fusion protein was expressed at the induction of IPTG.By affinity chromatography with glutathione sepharose 4B,GST hFAS fusion protein was purified.By thrombin cleavage and second affinity chromatography,GST protein was removed and recombinant human FAS protein was purified.Recombinant GST FAS and purified FAS extracellular protein could be recognized by anti hFAS monoclonal antibody sc 714 G.With purified FAS as antigen,anti FAS polyclonal antibodies were prepared.Anti FAS Ab could induce apoptosis of U937 cells.