【摘要】 目的为探索参与调控结核分枝杆菌(MTB)持续性感染的相关分子,对MTBH37Rv株编码Rv1494、Rv1495假想蛋白基因进行克隆、表达及Westernblotting鉴定。方法采用Bioedit、Dnaman软件及Pfam数据库对MTBH37Rv株Rv1494、Rv1495基因编码蛋白质的氨基酸组分、蛋白模块以及其与大肠杆菌E.coli的mazEF蛋白家族的同源性进行分析。以H37Rv株基因组为模板,分别对Rv1494、Rv1495基因进行克隆,构建pET32a(+)原核表达质粒,转化BL21宿主菌。重组蛋白经SDS-PAGE电泳分离纯化和Westernblotting鉴定。结果生物信息学分析提示,Rv1494基因、Rv1495基因编码的蛋白质分别与大肠杆菌E.coli染色体编码的mazEF家族中的抗毒素和毒素具有一定程度的同源性,分别为26%和29.75%;经测序表明原核表达重组质粒构建成功。负载重组质粒的BL21菌经IPTG诱导后可表达出分子大小与预期值相一致的融合蛋白,表达量均可达到菌体总蛋白的60%。重组蛋白经Westernblotting检测出特异性阳性信号。结论首次对MTBH37Rv株编码Rv1494、Rv1495假想蛋白基因进行克隆表达,为进一步探讨该基因在参与调控MTB持续性感染过程中的功能奠定基础。更多还原

【Abstract】 Objective The mechanism by which M.tuberculosis persists and survives in host macrophage is not fully understood, however, the M. tuberculosis chromosome-encoded TA loci perform functions possibly of signaling to these processes. To explore the biological functions of M. tuberculosis chromosome-encoded TA loci, the Rv1494 and Rv1495 genes of M.tuberculosis H37Rv strain were cloned and expressed. Methods The hypothetical proteins Rv1494 and Rv1495 were bioinformatically analyzed by means of Bioedit software, Dnaman software and Pfam database. The complete open-reading frame sequences of Rv1494 and Rv1495 genes were amplified by PCR using M.tuberculosis H37Rv genomic DNA as the template, and the PCR products were cloned into prokaryotic expression vector pET32a(+), respectively. After induction of expressions in E.coli host strain BL21 (DE3), the recombinant proteins were purified and detected by Western blotting. Results According to bioinformatic analysis, the hypothetical proteins of Rv1494 and Rv1495 genes shared some homologies with mazEF family, one of E. coli chromosomal TA loci (homology at 26% and 29.5%). Sequence analysis showed that the inserted target genes and its reading frames were completely correct. The recombinant plasmids were induced with IPTG to effectively express the fusion proteins with relative molecular mass coincident with prediction. The specific positive signals were identified from the immunoblots. Conclusion For the first time, the Rv1494 and Rv1495 genes of M.tuberculosis H37Rv strain were cloned and its prokaryotic expression vectors were constructed successfully in this experiment, which may facilitate further functional study of this mazEF-like gene pair.更多还原