毛冠鹿AIF-1原核载体的构建及表达  

Prokaryotic Expression System Construction of the Tufted Deer (Elaphodus cephalophus) AIF-1

作　　者：周楠楠[1] 杨振[1] 罗丽芸[1] 宋菲[1] 白敏[1] 曹祥荣[1] 

机构地区：[1]南京师范大学生命科学学院,江苏省分子医学生物技术重点实验室,江苏南京210023

出　　处：《南京师大学报（自然科学版）》2014年第2期85-90,95,共7页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金(30771172);国家基础科学人才培养基金(J1103507;J1210025)

摘　　要：从毛冠鹿睾丸cDNA文库中筛选出毛冠鹿AIF-1基因,对其进行生物信息学分析,设计引物克隆毛冠鹿AIF-1 cDNA,连入pMD19-T载体,测序正确后酶切,与表达载体pET-28a(+)连接,转化E.coli BL21(DE3),IPTG诱导表达,将诱导表达重组蛋白的菌体超声破碎后,进行可溶性分析,并对可溶性蛋白进行纯化,SDSPAGE电泳,Western Blot分析以及鉴定重组蛋白.结果表明,毛冠鹿AIF-1(TdAIF-1)含有1个438bp的开放阅读框,编码145个氨基酸,经测序和酶切鉴定后,成功构建重组质粒pET-28a(+)-TdAIF-1,表达大小约20 kD的重组蛋白,主要以可溶形式存在,提高洗脱缓冲液中咪唑浓度至50 mmol/L、100 mmol/L能够得到较纯的蛋白.成功构建毛冠鹿AIF-1原核表达体系,获得重组蛋白,为研究AIF-1蛋白的生物学功能奠定了基础.The TdAIF-1 cDNA was cloned from the testis cDNA library of the Tufted deer（ Elaphodus cephalophus） and analyzed by bioinformatic methods. Primers were designed according to cDNA sequence to clone the gene. The gene was cloned into pMD19-T vector for sequencing. The right sequence was digested by restriction enzyme and subcloned into the expression vector pET-28a（＋）. After transformed into E. coli BL21（DE3）,the recombinant plasimid was induced to express by IPTG. The E. coli BL21（DE3）expressed recombinant protein was broken by ultrasonic to show whether the recombinant protein was soluble or not. Lastly,the soluble protein was purified. The recombinant protein was analyzed and identificated by SDS-PAGE electrophoresis and Western blot. Analysis of sequence showed that the TdAIF-1 cDNA contained a 438 bp open reading frame encoding 145 amino acids. The recombinant plasimid was correctly constructed according to sequencing and restriction enzyme analysis. The recombinant protein was about 20 kD and soluble mainly. When the recombinant protein was purified,using elution buffer containing 50 mmol/L or 100 mmol/L imidazole could get purified protein. The TdAIF-1 Prokaryotic Expression System was constructed successfully and recombinant protein was obtained,which was helpful for the future study of its biological function.

关 键 词：毛冠鹿 AIF-1 原核表达 

分 类 号：Q28[生物学—细胞生物学]