牛趋化因子受体4基因的克隆表达及序列分析  

Cloning,expression and sequencing of bovine CXCR4 gene

作　　者：李小庆[1] 何延华[1] 陈国华[1] 房永祥[1] 冯海燕[1] 赵娜[1] 景志忠[1] 

机构地区：[1]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,甘肃兰州730046

出　　处：《中国兽医科学》2009年第11期1003-1009,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30871884);国家高技术研究发展计划(863)项目(2006AA10A203)

摘　　要：采用RT-PCR技术,从被刺激诱导的牛外周血单个核细胞（PBMC）中克隆了牛趋化因子受体4（CXCR4）全长基因,并将CXCR4基因和CXCR4基因N端1~126 bp（CXCR442 aa）片段分别亚克隆到原核表达载体pGEX-4T-1中,命名为pGEX-4T-1-CXCR4、pGEX-4T-1-CXCR442 aa。获得重组菌株后,采用不同的IPTG浓度、不同诱导时间、不同温度和不同培养基等组合诱导表达这两种重组菌,其中pGEX-4T-1-CX-CR442 aa重组菌获得了表达,并在37℃、IPTG浓度为0.8 mmol/L、诱导6 h时表达量最大,约占菌体总蛋白的45%,目的蛋白主要以包涵体的形式存在;而pGEX-4T-1-CXCR4重组菌未表达出目的蛋白。A full-length chemokine receptor 4（CXCR4） gene was amplified by RT-PCR from total RNA of bovine peripheral blood mononuclear cells（PBMC） stimulated with LPS and ConA,then cloned and sequenced.The subcloned full-length CXCR4 gene and a fragment of 126bp in size at N-terminal（CXCR442 aa） were successfully inserted into the expression vector pGEX-4T-1,named by pGEX-4T-1-CXCR4 and pGEX-4T-1-CXCR442 aa,respectively.Recombinant Escherichia coli BL21 was induced with different concentration of IPTG to express recombinant protein.SDS-PAGE analysis showed that the optimal temperature,IPTG concentration,and induction time for the recombinant pGEX-4T-1-CXCR442 aa were 37℃,0.8mmol/L,and 6 hours,respectively,and the recombinant protein existed mainly in the form of inclusion bodies.The fusion protein,approximately 31ku in molecular weight,almost made up 45% of the total bacterial proteins.However,the recombinant protein was not expressed in pGEX-4T-1-CXCR4 even in various temperatures,concentrations of IPTG,induction time and media.

关 键 词：牛 CXCR4基因 克隆 表达 结构预测 

分 类 号：S852.42[农业科学—基础兽医学]