牛分枝杆菌ESAT-6基因和CFP-10基因在大肠杆菌中的融合表达

Fusion expression of ESAT-6 gene with CFP-10 gene of Mycobacterium bovis in Escherichia coli

以牛分枝杆菌Vallee株基因组DNA为模板,应用PCR扩增获得了ESAT-6和CFP-10两个目的基因片段,采用重叠延伸剪接技术(SOE)将ESAT-6基因和CFP-10基因通过(G1y4Ser)3接头连接,得到了融合基因。将融合基因片段先克隆于pMD18-T载体,再亚克隆到表达载体pET32a(+)中,通过限制性内切酶消化、菌液PCR鉴定、序列分析证实得到含预期序列的重组质粒pET-ESAT-6-CFP-10;该质粒转化BL21(DE3);经IPTG诱导,表达出了40ku的融合蛋白;用Ni螯合层析方法纯化该蛋白,经SDS-PAGE检测,出现了清晰的单一条带;Western-blotting试验结果显示,该融合蛋白能与抗牛分枝杆菌阳性血清发生反应。结果表明,ESAT-6基因和CFP-10基因在大肠杆菌中融合表达成功并具有良好的反应原性。

ESAT-6 gene and CFP-10 gene were amplified by PCR from genomic DNA of Mycobacterium boris Vallee strain. By gene SOEing method, the ESAT-6 gene and CFP-10 gene of M. boris were linked with the（Gly4 Ser）3 linker. The linked ESAT-6-CFP-10 gene was inserted initially into pMD18-T vector,and then sub-cloned into the expression vector pET32a（＋）. The recombinant plasmid pET-ESAT- 6-CFP-10,which was confirmed to be correct by digestion with restriction endonuclease,PCR amplification and sequencing,was transformed into Escherichia coli BL21 （DE3）. The bacteria was induced by IPTG and the expressed protein was purified by affinity chromatography. Analyses by SDS-PAGE and Western-blot tests revealed that the fusion protein 40 ku in size was expressed in the BL21（DE3） and one distinct band was presented in SDS-PAGE. The purified protein could be recognized by positive sera of cattle infected with M. boris in Western-blot test. It was concluded that the ESAT-6-CFP-10 gene was expressed successfully in E. coli and the expressed fusion protein had excellent antigenicity.