牛分枝杆菌抗原MPB70、MPB83和ESAT-6的融合表达及重组蛋白的初步应用

应用PCR方法从牛分枝杆菌Vallee株基因组中扩增获得mpb70、mpb83和esat-6三个目的基因片段。采用重叠延伸剪接技术(splicing by overlap extension,SOE)获得融合基因mpb70-mpb83后,将mpb70-mpb83和esat-6串连于同一表达载体pET32a( )中得到重组质粒pET70-83-E6。转化BL21(DE3)大肠杆菌感受态后,经IPTG诱导以可溶的形式表达融合蛋白。用Ni2 亲合层析的方法纯化该融合蛋白。Western blot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,而与牛副结核病阳性血清不反应。用该纯化蛋白初步建立了间接ELISA方法,并检测了117份临床血清样本(其中67份为PPD阳性牛血清),阳性率为39.32%(46/117)份,与PPD皮试诊断的符合率为82.05%(96/117)。

Fusion Expression of MPB70, MPB83 and ESAT-6 of Mycobacterium Bovis and Its Use as Diagnostic Reagent

The DNA fragments of mpb70,mpb83 and esat-6 were amplified by PCR from M.bovis Vallee genome DNA.Mpb70 and mpb83 were spliced by overlapping extension(SOE).Then the DNA fragments were inserted into the plasmid pET32a( ) and the recombinant plasmid pET70-83-E6 was accquired.The recombinant protein was expressed in the form of solution in pET70-83-E6 transformed BL21(DE3)induced by IPTG.Then the recombinant protein was purified by affinity chromatography.Western blot analysis shows that the protein could be recognized by sera from M.bovis infected bovines but not by sera from M.paratuberculosis infected bovines.The purified protein was used as diagnostic reagent and found to be useful for immunodiagnostic of bovine tuberculosis(BTB) by enzyme linked immunosorbent(ELISA).In field test,46 out of 117 bovine sera(67 of them were positive in PPD skin test) were positive(39.32%).82.05%((96/117)) of them were consistent with PPD skin test.