结核分枝杆菌Rv0859基因的克隆、表达、纯化及蛋白的亚细胞定位

Cloning and Expression of Mycobacterium tuberculosis Rv0859 Gene and Its Purification

摘要:

本研究体外克隆了结核分枝杆菌Rv0859基因, 融合表达并纯化了Rv0859蛋白。首先提取H37Rv标准菌株中的基因组DNA, 设计Rv0859基因两端的引物, 以H37Rv基因组DNA为模板通过PCR方法扩增Rv0859基因。用Hind III和BamHⅠ两种限制性内切酶双切Rv0859基因, T4连接酶连接到pET30载体上, 再转入大肠杆菌JF1125中, 经过筛选鉴定后抽提质粒测序, 得到重组正确载体, 转化到表达宿主大肠杆菌BL21中。用IPTG进行诱导表达, 通过聚丙酰胺凝胶电泳(SDS-PAGE)及质谱鉴定重组表达蛋白。0.05 mol/L浓度的IPTG 37°C诱导4 h重组蛋白的表达量最高。制备重组蛋白的多克隆抗体, 通过亚细胞分离及Western-blotting分析蛋白的亚细胞定位。结果成功地构建原核表达载体pET30a-Rv0859, 并获得47845 D左右的大量表达的Rv0859蛋白, Western-blotting结果表明Rv0859蛋白主要定位于细胞膜中, 微量存在于细胞壁中, 为进一步的Rv0859蛋白功能研究奠定了一定的基础。

Abstract:

To clone Rv0859 gene of Mycobacterium tuberculosis and purify its fusion protein. Genome DNAs of H37Rv was extracted. Primers for Rv0859 gene was designed, and DNA fragments encoding Rv0859was obtained by PCR and inserted into proeukaryotic expression plasmid pET30a followed by digestion with Hind III and BamH I. pET30a-Rv0859 is connected by T4 ligase which was transformed into Escherichia coli JF1125. Sequence analysis was performed to verify the recombinant plasmid. The recombinant plasmid was transfected into Escherichia coli BL21. After induction with IPTG, the expression of Rv0859 protein was identified by SDS-PAGE and mass spectrum. The expressed protein was the maximum when induced with 0.05 mol/L IPTG for 4 h at 37°C. The polyclonal antibodies were prepared to detect the subcellular localization by Western-blotting. The result is the prokaryotic expression vector pET30a-Rv0859 was constructed successfully and the recombinant Rv0859 was expressed heavily. The result of Western-blotting indicated that the protein located on the cell envelope partly and little on the cell well. The study laid the foundation for application in the tuberculosis.