牛分枝杆菌抗原MPB70、MPB83、CFP-10和ESAT-6的融合表达及相关特性分析

目的牛分枝杆菌抗原MPB70、MPB83、CFP-10和ESAT-6的融合表达及相关特性分析。方法应用PCR方法从牛分枝杆菌临床分离株基因组中扩增获得mpb70、mpb83、cfp-10和esat-6四个目的基因片段。采用重叠延伸剪接技术(splicing by overlapextension,SOE)获得融合基因cfp10-esat6和mpb83-cfp10-esat6,将mpb70和mpb83-cfp10-esat6串连于载体pUC19-Linker上,再将mpb70-mpb83-cfp10-esat6连于表达载体pET28a(+)中得到重组质粒pET70-83-C10-E6。转化BL21(DE3)感受态细胞后,经IPTG诱导获得以可溶形式表达的融合蛋白。用Ni2+亲合层析法纯化该融合蛋白。结果经ELISA验证该融合蛋白能分别与抗原蛋白MPB70、MPB83和CE(cfp10-esat6)的多抗反应,说明该融合蛋白具有四个抗原蛋白的免疫学活性。Westernblot分析显示:该融合蛋白能与抗牛分枝杆菌阳性血清发生特异性反应,而与牛其它疾病的阳性血清不反应。热稳定性试验证明该融合蛋白属于热稳定性蛋白。结论融合表达了牛分枝杆菌的四种特异性抗原蛋白,该蛋白具有单个蛋白的免疫原性和稳定性,作为一种新型的诊断抗原具有良好的应用前景。

Fusion expression of MPB70,MPB83,CFP-10 and ESAT-6 antigens from Mycobacterium bovis and analysis of their related properties

To investigate the fusion expression of MPB70,MPB93,CFP-10 and ESAT-6 antigens from Mycobacterium bovis and to analyze their related properties,the DNA fragments of 4 target genes mpb70,mpb83,cfp-10 and esat-6 were amplified from genomic DNA of M bovis isolates.The fusion genes cfp-10-esat-6 and mpb83-cfp-10-esat-6 were obtained through the splicing of overlapping extension technique.The gene mpb70 and fusion gene mpb83-cfp10-esat-6 were first inserted to the plasmid pUC-19 linker,and then the fusion gene mpb83-cfp-10-esat-6 were inserted to the expression plasmid pET32a(+),thus to obtain the resultant recombinant plasmid pET70-83-C10-E6.The fusion protein was expressed by transformation of pET70-83-C10-E6 into BL21(DE3)cells after IPTG induction and the recombinant soluble protein was purified with Ni2+ affinity chromatography.As demonstrated by ELISA assay,the expressed protein could be recognized by positive antiserum to M bovia and the mono-specific antisera against MPB70,MPB83,CFP-10 and ESAT-6 antigens.As demonstrated by Western blotting,this protein could be identified only by antiserum against M bovis,but not by antisera against M.paratuberculosis,Brucella,Babesia and infectious bovine rhinotracheitis virus.Heat treatment test showed that the fusion protein was heat-stable at 60℃ for one hour.It is concluded that 4 specific antigenic fusion proteins are obtained through fusion expression of MPB70,MPB83,CFP-10 and ESAT-6 antigens from M.bovis,and they can be used as the diagnostic antigen to detect the presence of the related infection.