结核分枝杆菌Rv3803c、Rv3846基因的克隆、表达、纯化与鉴定

Cloning,expression,purification and identification of Rv3803c,Rv3846 gene from Mycobacterium tuberculosis

目的克隆结核分枝杆菌Rv3803c和Rv3846基因,并在大肠埃希菌(E.coli)中进行表达和纯化。方法以结核分枝杆菌H37Rv基因组DNA为模板,应用PCR方法扩增出Rv3803c和Rv3846基因片段,克隆至pGEX-6P-1表达载体中,测序正确后,在E.coli Rosetta中诱导表达,GST标签亲和层析法纯化蛋白,考马斯亮蓝染色及Western-blot分析鉴定。结果重组质粒pGEX-Rv3803c、pGEX-Rv3846测序表明核苷酸序列与设计完全一致。其在E.coli Rosetta中的存在形式均为可溶部分约50%、包涵体约50%,相对分子质量约为56 000和45 000,纯化的GST-Rv3803c、GST-Rv3846样品纯度90%以上。完整的GST-Rv3803c的浓度大约为0.1mg/ml,完整的GST-Rv3846的浓度大约为0.5mg/ml。即相当于每升E.coli Rosetta培养物中,GST-Rv3803c产量为:0.25mg/L、GST-Rv3846产量为1.25mg/L。结论成功获得了纯化蛋白Rv3803c(MPT51)和Rv3846(SodA),为进一步研究MPT-51和SodA蛋白的辅助诊断价值、构建保护性疫苗提供了实验依据。

Objective To clone and express the genes Rv3803c and Rv3846 from Mycobacterial tuberculosis in Escherichia coli cells,and to purify Rv3803c and Rv3846 proteins.Methods The genes Rv3803c and Rv3846 were amplified from Mycobacterial tuberculosis H37Rv genomic DNA by polymerase chain reaction（PCR）.PCR products were cloned into the expression vector pGEX-6P-1.After the plasmids were sequenced and validated their correctness,the recombinant plasmids were transformed into E.coli Rosetta strain to induce the expression of proteins.The proteins were purified by the GST-tag affinity chromatography,and then electrophoresed,stained with Coomassic brilliant blue,identified by Western-blot.Results The sequences of recombinant plasmids pGEX-Rv3803c and pGEX-Rv3846 are consistent with the design.The proteins GST-Rv3803c and GST-Rv3846 existed 50% in the soluble fraction and 50% in the inclusion body of E.coli Rosetta.Their molecular weights were 56 000 and 45 000,respectively.The purities of final products were higher than 90%.The concentrations of the purified recombinant proteins were approximately 0.1 mg/ml and 0.5 mg/ml,respectively.The yields of the recombinant proteins GST-Rv3803c and GST-Rv3846 were 0.25 mg and 1.25 mg from per liter of the E.coli Rosetta cultures,respectively.Conclusion The purified proteins Rv3803c（MPT51）and Rv3846（SodA）were successfully obtained,which provide the experimental basis for the further study of auxiliary diagnostic value of MPT51 and SodA proteins and construction of protective vaccines.