兔视黄醇结合蛋白4基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Rabbit Retinol Binding Protein 4 Gene

作　　者：王泽祥[1] 孙宏勇[1] 郭东春[1] 单丽玲[1] 原冬伟[1] 刘家森[1] 曲连东[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/实验动物研究室,黑龙江哈尔滨150001

出　　处：《动物医学进展》2013年第7期53-56,共4页Progress In Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费专项(0302012008)

摘　　要：为表达兔视黄醇结合蛋白4(rRBP4),提取兔肝脏组织总RNA,采用RT-PCR技术扩增了rRBP4的基因,大小为606bp。将其克隆于原核表达载体pET-32a(+)获得表达重组质粒,并转化大肠埃希菌BL21(DE3)Plyss中进行表达,pET-32a-rRBP4以包涵体形式表达,包涵体经过洗涤、变性后,用镍离子亲和层析柱纯化,并利用尿素梯度透析法复性。SDS-PAGE分析表明,获得与预期分子质量一致的特异性目的蛋白;Western blot检测表明,重组蛋白与小鼠抗His标签单克隆抗体发生特异性反应。To prokaryotic express the rabbit retinol binding protein 4,total RNA was isolated from nomal rabbit liver,then RBP4gene was amplified by RT-PCR and inserted into pET-32a（＋）vector for expression in E.coli BL21（DE3）Plyss.SDS-PAGE analysis showed that the recombinant protein was expressed after induction with 1mmol/L IPTG at 37℃.The recombinant fusion protein,which was forms inclusion in body,was washed,denatured,and purified by Ni-NTA agarose affinity column and renatured by dialysis.Western blot indentification showed the recombinant protein was recognized by MAb anti-His tag.The induced and purified protein rRBP4could be used for further study of interaction with rabbit hemorrhagic disease virus.

关 键 词：兔视黄醇结合蛋白4 原核表达 肝细胞 

分 类 号：Q785[生物学—分子生物学]