【摘要】 目的对人趋化因子受体CCR5的N端胞外部分与第二个胞外环(extracellular loop-2,ECL-2)进行融合表达与纯化。方法分别扩增人趋化因子受体CCR5的胞外N-端部分与ECL-2部分相应的编码序列,通过重叠延伸拼接(Splicing byoverlapping extension,SOE)PCR实现两片段DNA的拼接后(命名为CCR5-N-E2)克隆入质粒pBlueScript M13中,同时构建原核表达载体pET-21b(+)-CCR5-N-E2,转入表达菌株BL21(DE3),IPTG诱导表达重组蛋白CCR5-N-E2,表达产物通过蛋白免疫印迹进行鉴定,并采用金属螯合层析法对重组表达产物进行纯化。结果经测序,构建的重组原核表达载体与预期完全一致,相对分子质量为8 500的目的蛋白在BL21(DE3)菌株中得到高效表达,表达量约占总蛋白的50%,表达产物以包涵体形式存在。蛋白免疫印迹实验表明该重组蛋白与抗CCR5 N-端序列的单克隆抗体发生特异性结合。通过Ni2+亲和层析,目的蛋白的纯度可达95%以上。结论实现CCR5-N-E2编码序列的拼接、融合蛋白的表达以及纯化,为广泛筛选以CCR5为靶点的临床治疗药物奠定了基础。更多还原

【Abstract】 Objective To express and purify the first extracellular domain and extracellular loop 2(ECL-2) of human chemokine receptor CCR5.Methods Gene fragments of the first extracellular domain and ECL-2 were amplified respectively and then spliced by Splicing of overlapping extension(SOE) PCR.This fragment was inserted into the prokaryotic expression vector pET-21b(+) and transformed into E.coli strain BL21(DE3),and then inducted with IPTG.The recombinant protein fusing with 6His-tag,named CCR5-N-E2,was detected by Western blot analysis.Metal-chelating affinity chromatography was performed to purify CCR5-N-E2.Results SDS-PAGE analysis showed that the recombinant protein(Mr 8 500) was expressed in E.coli in the form of inclusion body at the expression level over 50% of the total cell protein.After metal-chelating affinity chromatography,the recombinant protein reached the purity over 95%.Result of Western blotting showed that the recombinant protein could react with anti-human CCR5 antibody.Conclusion Recombinant protein including the first extracellular domain and ECL-2 of human chemokine receptor CCR5 is expressed and highly purified,which will facilitate the study of inhibitor agents against HIV-1 targeting CCR5.更多还原