表达结核分枝杆菌休眠相关抗原Rv2031c-Rv2626c融合蛋白重组耻垢分枝杆菌的构建与鉴定

Construction and Identification of Recombinant Mycobacterium smegmatis Expressing the Fusion Protein of Dormancy Antigens Rv2031c and Rv2626c of Mycobacterium tuberculosis

利用大肠埃希菌—分枝杆菌穿梭表达载体pDE22,构建能够表达结核分枝杆菌(Mycobacterium tuberculosis,MTB)休眠相关抗原Rv2031c-Rv2626c融合蛋白的重组耻垢分枝杆菌(Mycobacterium smegmatis,M.s)。采用聚合酶链反应(PCR)方法,从MTB H37Rv基因组DNA中扩增MTB休眠相关抗原Rv2031c和Rv2626c基因片段并克隆入pMD18-T载体。序列测定正确后分别亚克隆入大肠埃希菌—分枝杆菌穿梭表达载体pDE22。将重组载体pDE22-Rv2031c-Rv2626c电穿孔导入M.s,42℃热诱导表达目的蛋白,并进行SDS-PAGE和Western-blot分析。利用PCR方法扩增获得MTB休眠相关抗原Rv2031c和Rv2626c基因片段,大小分别为435 bp和480 bp。基因测序与Genebank公布的基因序列完全一致。SDS-PAGE分析结果表明重组载体pDE22-Rv2031c-Rv2626c电穿导入M.s后能够特异性表达目的蛋白,大小约为35 kDa,与理论值相符。Western-blot鉴定结果表明抗Rv2031c和抗Rv2626c的单克隆抗体均能够与目的蛋白发生特异性的反应,大小相一致。成功构建表达MTB休眠相关抗原Rv2031c-Rv2626c融合蛋白的重组M.s,并命名为Rv2031c-Rv2626c-rM.s,为其用于结核新型疫苗的研究奠定了基础。

To construct the recombinant Mycobacterium smegmatis（M.s） expressing the fusion protein of dormancy antigens Rv2031c and Rv2626c from Mycobacterium tuberculosis（MTB） using an E.coli-Mycobacterium shuttle expression vector.Rv2031c and Rv2626c genes was amplified from MTB H37Rv genome DNA by PCR and sequenced in the pMD18-T vector.The fusion gene of Rv2031c-Rv2626c was connected in the pMD18-T vector using enzyme restriction sites and then subcloned into the E.coli-mycobacterium shuttle vector pDE22.The recombinant vector pDE22-Rv2031c-Rv2626c was transformed into M.s by electroporation.The recombinant M.s was analyzed the gene type by PCR,then induced at 42 °C,to analyze the expression of fusion protein Rv2031c-Rv2626c by SDS-PAGE and Western-blot.The sizes of Rv2031c and Rv2626c gene fragments were 435 bp and 480 bp seperately,and the sequences were identical to those of genes in the GenBank.The fusion gene fragment of Rv2031c-Rv2626c could be amplified from the recombinant M.s by PCR.The reslult of SDS-PAGE showed that the fusion protein Rv2031c-Rv2626c could be specifically expressed in M.s,with a relative molecular mass of 35 kDa.The Western-blot showed that the fusion protein Rv2031c-Rv2626c could be detected by the monoclonly antibodies of Rv2031c and Rv2626c,and there is a specific binding band appearred at the corresponding site.In conclusion,the recombinant M.s expressing the fusion protein Rv2031c-Rv2626c is successfully constructed,and named as Rv2031c-Rv2626c-rM.s.The value of the rM.s as a TB candidate vaccine will be studied.