斑马鱼p53基因原核表达载体的构建及其在大肠杆菌中的表达  

Prokaryotic Vector Construction and Expression of Zebra Fish p53 Gene

作　　者：赵向忠[1,2] 刘明[1] 丁丽丽[1] 吴宁[1] 林秀坤[1] 

机构地区：[1]中国科学院海洋研究所,山东青岛266071 [2]中国科学院研究生院,北京100039

出　　处：《湖北农业科学》2010年第3期526-528,532,共4页Hubei Agricultural Sciences

基　　金：国家自然科学基金(30472043);山东省科技攻关项目(2004C07)

摘　　要：利用原核表达系统克隆表达斑马鱼p53基因。RT-PCR法从斑马鱼胚胎中扩增获得p53基因编码区,并将其克隆至原核表达载体pET28a上,构建重组质粒pET28a/z-p53,将重组质粒转化E.coli BL21(DE3)受体菌,IPTG诱导表达,表达产物经镍柱纯化、尿素透析复性,SDS-PAGE电泳分析,结果表明,p53基因在大肠杆菌中成功表达,表达的p53融合蛋白分子量大约为53kD,透析复性后获得了高纯度可溶性的p53蛋白。The cloning and expressing of zebra fish p53 gene were studied by using prokaryotic expression system.p53 encoding region was obtained from zebra fish embryos by RT-PCR,and then the p53 fragment was cloned in pET28a vector to construct the recombinant plasmid pET28 a / z-p53.It was transformed into E.coli BL21,and then induced by 1mmol/L IPTG to express p53 protein in E.coli BL21 expression system.The recombinant p53 protein was purified by Ni-NTA affinity chromatography under denaturing conditions and renatured through urea gradient dialysis.Soluble p53 protein with high purity was successful obtained.

关 键 词：P53基因 斑马鱼 原核表达 

分 类 号：Q786[生物学—分子生物学]