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

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.