人凋亡相关基因TFAR19缺失突变体的原核表达、产物纯化及鉴定

构建TF 1细胞凋亡相关基因 19(TF 1cellapoptosisrelatedgene 19,TFAR19)缺失突变体的原核表达载体 ,获取缺失突变体蛋白 ,用于TFAR19促凋亡分子机理的研究 .从真核表达载体pcDI TFAR19扩增出野生型TFAR19和 4个缺失突变体 ,重组到原核表达载体pGEX 4T 2 .经亲和层析方法对缺失体蛋白进行纯化后 ,再利用凝胶过滤的方法进一步纯化 .利用抗GST和抗TFAR19的单克隆抗体对蛋白进行免疫学鉴定 .用白血病细胞株HL 6 0检测蛋白活性 .成功地克隆并重组了野生型TFAR19及缺失突变体 pGEX 4T 2表达载体 ,对融合蛋白的表达条件进行了优化 .SDS PAGE结果显示 ,各个缺失突变体融合蛋白均有较高水平的表达 .免疫学检测证实获得了正确的表达产物 .活性检测证实 ,野生型TFAR19和缺失突变体 4可以明显促进去血清诱导的HL 6 0细胞凋亡 ,第 6外显子可能是一个与TFAR19促凋亡活性密切相关的功能结构域

Expression,Purification and Identification of Deletants of Human TF-1 Cell Apoptosis Related Gene-19 ( TFAR 19) in E.coli

To express wtTFAR19 and its deletants in E. coli and study the enhancing apoptosis mechanism by TFAR19, the cDNA encoding wtTFAR19 and different deletants were amplified and cloned into pGEX 4T 2. The recombinant proteins were expressed in E.coli BL21 and the inducing conditions were optimized, and then purified by Glutathione Sepharose TM 4B affinity chromatography and gel filtration. The anti GST and anti TFAR19 monoclonal antibodies were used for identification of the recombinant proteins. The purified proteins were co cultured with HL 60 cells and the apoptotic cells were analyzed by PI and Annexin V labeled FACS. SDS PAGE analysis showed high level expression of deletant proteins. Western blot results showed that the right recombinant protein was obtained. The FACS results showed that wtTFAR19 and deletant 4 enhanced the apoptosis of HL 60 cells induced by deprivation of serum. It has showed that recombinant protein of TFAR19 deletants was obtained and had the enhancing apoptosis activity. The exon 6 could facilitate the enhancing apoptosis activity of TFAR19.