人Fas cDNA的克隆及其在大肠杆菌中表达的研究

CLONING AND EXPRESSING OF HUMAN FAS cDNA IN E.coli

为获得高质量及充足的Ｆａｓ蛋白，采用ＰＣＲ技术调整Ｆａｓ基因的开放阅读框架，使之与生物素化蛋白基因阅读框架一致；缺失了ＦａｓｃＤＮＡ基因的起始密码子并增加一个大肠杆菌偏性终止密码子，构建ＦａｓｃＤＮＡ和生物素化融合原核表达质粒ＰｉｎＰｏｉｎｔ－Ｆａｓ。将重组质粒转入大肠杆菌ＨＢ１０１，经５００ｍｍｏｌＩＰＴＧ在３７℃条件下诱导４ｈ，ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ印迹检测融合蛋白在大肠杆菌得以高效表达，表达量为细菌总蛋白的１３．８％。用亲和层析树脂对生物素化融合蛋白进行亲和层析纯化，得到Ｆａｓ重组的蛋白，且表达的Ｆａｓ融合蛋白具有抗体结合活性。此蛋白的表达成功将解决Ｆａｓ膜蛋白不易提取的难题。

For the purpose of obtainning sufficient quantity of Fas protein, we constructed E. coli expressed vector PinPoint Fas, and Fas cDNA fused to the 3’ end of the gene encoding the Bitin hinding protein with its own signal sequence. By PCR we made Fas cDNA coding sequence which has the same open reading frame as Biotin hinding protein gene, meanwhile defected initiating methionine codon of the oviqinal Fas cDNA and added a terminator that E. coli favor. The fusion protein was expressed in E, coli HB 101 at 37℃after 500mmol IPTG induction for 4h. The fusion protein of high expression has a molecular weight of 33.4kDa by SDS PAGE,western blotting analysis showed Fas fusion protein maitained immunoveactivity with Fas McAb. The yield of the Fas fused protein was 13.8 percent in total protein. Using avidin resin, we affinity purified the biotinylated fusion proteins under nondenaturing couditions. The successful expression of Fas fusion protein will provide sufficient material for further suelies of Fas.