秦川牛天然免疫力相关巨噬细胞蛋白1基因(Nramp1)N端序列的原核表达及纯化  

Prokaryotic Expression and Purification of Qinchuan Cattle's Natural Resistance-associated Macrophage Protein-1 Gene(Nramp1) N-terminal Fragment

作　　者：李倩[1] 徐晓彬[1] 陈华涛[1] 王爱华[1] 张涌[1] 靳亚平[1] 马延森[1] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100

出　　处：《农业生物技术学报》2011年第1期142-148,共7页Journal of Agricultural Biotechnology

基　　金：国家重大课题(No.2008ZX08007-004);陕西省重大专项(No.2008ZDKG-05)资助

摘　　要：为了研究牛天然免疫力相关巨噬细胞蛋白1(Nramp1)的功能,探求转基因抗胞内感染菌牛新材料创制的功能基因,本实验应用RT-PCR方法从秦川牛(Bos taurus)外周血中扩增出Nramp1编码基因N端序列,并将其克隆到pMD18-T simple载体,酶切后连接于pGEX-4T-1表达载体,命名为pGEX-N2。将重组质粒pGEX-N2转化大肠杆菌(Escherichia coli)BL21(DE3),经IPTG诱导,SDS-PAGE电泳检测GST-Nramp1-N融合蛋白表达,并筛选最佳诱导条件,用谷胱甘肽Sepharose4B介质分离纯化该融合蛋白。结果表明,牛Nramp1N端编码序列长186bp,与GenBank中的相应的牛基因序列(U12862.1)对比存在一个碱基差异,致使其编码的第49位氨基酸为丙氨酸。构建的牛Nramp1N端表达重组质粒pGEX-N2,于35℃、0.1mmol/LIPTG、诱导10h,在大肠杆菌中高效表达,表达产物的分子量为33kD。结果为进一步研究牛Nramp1在机体抵抗胞内菌感染中的作用并为利用该基因进行转基因抗病牛的培育提供了实验依据和实验材料。To explore the function of bovine natural resistance-associated macrophage protein-1（Nramp1） and seek for the objective genes for resistance breeding against intracellular pathogen via transgenic technology,Nramp1 gene N-terminal fragment was amplified from total RNA of Qinchuan cattle＇s（Bos taurus） peripheral blood by RT-PCR,inserted into pMD18-T simple vector,and secondly subcloned into pGEX-4T-1 vector（named pGEX-N2）. And the recombinant plasmid pGEX-N2 was transformed into Escherichia coli BL21（DE3） strain. SDS-PAGE analysis showed that GST-Nramp1-N fusion protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. Eventually,GST-Nramp1-N fusion protein was purified by glutathione Sepharose 4B. The results demonstrated that Qinchuan Cattle＇s Nramp1 gene N-terminal fragment was 186 bp; Compared to gene order of Nramp1 published at GenBank（U12862.1）,it had a single nucleotide mutation. This nucleotide mutation made the 49th amino acid which encoded by the Qinchuan cattle＇s Nramp1 gene N-terminal fragment changed to the alanine. Recombinant plasmid pGEX-N2 was expressed efficiently in the Escherichia coli BL21（DE3） under the optimized induction condition （0.1 mmol/mL IPTG for 10 h at 35℃） and its expressive product （33 kD） was soluble; In brief,the findings that depurated GST-Nramp1-N fusion protein obtained in the present study provides a good foundation for further research to determine the biological activity of bovine Nramp1 and lay a pathway for resistance breeding by animal transgenic technology.

关 键 词：天然免疫力相关巨噬细胞蛋白基因1(Nramp1) 原核表达 蛋白纯化 秦川牛 

分 类 号：S567.239[农业科学—中草药栽培]