猪CD8α分子胞外区基因片段的原核表达  

Prokaryotic expression of porcine CD8α extracellular domain

作　　者：崔青[1,2] 孙晓林[1] 王生富[2] 房永祥[2] 陈国华[2] 曾爽[2] 景志忠[1,2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点实验室,甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2010年第6期18-22,共5页Journal of Gansu Agricultural University

基　　金：国家自然基金项目(30871884);甘肃省农业生物技术专项(GNSW-2008-2);甘肃省科技支撑计划(0804NKCA076)

摘　　要：采用PCR技术从pGEM-pCD8α重组质粒中克隆了pCD8α胞外区去信号肽基因片段,并构建原核表达载体pET-30a-pCD8α-ED,在不同IPTG浓度和时间下进行诱导表达.结果表明:SDS-PAGE表达出约25ku的融合蛋白,目的蛋白主要以包涵体的形式存在,IPTG最佳诱导浓度为0.5mmoL·L-1,诱导6h时表达量达到峰值;Western-blot结果显示,在25ku左右出现杂交条带,表明表达的融合蛋白能被抗HIS标签的单克隆抗体识别,说明目的蛋白在大肠杆菌中得到了成功表达.In order to preparing more porcine CD8α-ED recombinant protein with biological activity,porcine CD8α molecule extracellular domain fragment（pCD8α-ED）was amplified by PCR method from the pGEM-T-pCD8α recombinant plasmid,the pCD8α-ED was cloned into pET-30a vector and expressed in Escherichia coli.Inducible expression was carried out in different concentrations of the IPTG and different inducible expression time.The results showed that the fusion protein was exists mainly in the form of inclusion bodies and with the molecular weight of 25 ku by SDS-PAGE analysis.The optimum inducible concentration of IPTG was 0.5 mmoL·L-1,and the optimum inducible time was 6 h.The analysis of Western-blot indicated that the hybridization band was appeared around in the 25 ku,and it showed that the fusion protein was identified by the monoclonal antibody of HIS label protein,so it was showed that the target protein was expressed successfully in the Escherichia coli.

关 键 词：猪CD8α分子 胞外区基因片段 原核表达 

分 类 号：S852.4[农业科学—基础兽医学]