The Expression Of Mycobacterium Bovis Fusion Gene Cfp10-esat6 And The Preliminary Application Of The Fusion Protein

Bovine tuberculosis(BTB),caused by Mycobacteriumbovis,is a zoonotic disease.Traditional diagnosis of bovine tuberculosis relies on tuberculin skin test,which suffers major limitation of low specificity due to cross-reactivity components in purified protein derivative of tuberculin (PPD).ESAT6 and CFP10,whose encoding genes existent in the genome of M.bovis but absent in that of M.avium and M.paratuberculosis,have considerable potential as diagnosis antigens for BTB.The DNA fragments of cfp10 and esat6 were amplified by PCR from M.bovis strain C68001.The fusion gene cfp10-esat6 was obtained via technique of splicing by overlapping extension(SOE).The fusion gene was cloned into pET32a(+) plasmid and named as pET32a(+)-cfp10-esat6.The recombinant plasmid was transformed into E.coli BL21(DE3) and expressed a soluble protein.This recombinant protein could be recognized by serum of M.bovis infected cattle in western blot.The fusion protein CFP10-ESAT6 was highly expressed and amounted to 42%of total bacterial protein under optimal inducing conditions.The purity of fusion protein was up to 90%after Ni-NTA purification.The specificity of the purified recombinant protein was assayed by skin test in the gnuinea pigs sensitized with M.bovis,M.avium,M.paratuberculosis,respectively.Fusion protein of 1μg can induce DTH responses of guinea pigs sensitized with M.bovis.1μg fusion protein corresponds to 10IU PPD.The weak DTH responses against recombinant protein was observed in the gnuinea pigs sensitized with M.avium or M.paratuberculosis.The results indicated that the recombinant protein can differentiate M.bovis from M.avium and M.paratuberculosis in sensitized guinea pigs.The skin tests were assayed in parallel with the purified recombinant protein and PPD in 40 cattles,the results indicated that recombinant protein of 16μg can induce strong DTH responses in M.bovis infected cattle,and the coincidence rate of the results between them was 85%.The recombinant protein and PPD were applied to IFN-γELISA test,respectively,recombinant protein of 2μg can induce IFN-γrelease,and the coincidence rate of the results between them was 90%.In conclusion,the purified recombinant protein has high sensitivity in both skin test and IFN-γELISA for clinic diagnosis,and has a promising application as an alternative antigen of PPD in diagnosis of BTB.