Expression And Purification Of Esat-6 And Mpt83 Proteins Of Bovine Tuberculosis And Their Primary Application In Elisa Detection

Bovine tuberculosis (TB) is a zoonotic disease of animals caused by Mycobacterium bovis, which threatens the health of human and animals, and result in hunge economic losses. In the present study, we selected two genes encoding secretory proteins from the complete genome. ESAT-6 and MPt83 are lipoprotein secreted in the supernatants when Mycobacterium bovis is cultured in the early period, which are important for investigating the seropreverlent and developing the vaccine of bovine TB.Two pairs of primers, which contain restriction enzyme sites, were designed according to the ESAT-6 sequence and MPT83 sequence in GenBank. The gene coding ESAT-6 and MPT83 protein were amplified by polymerase chain reaction (PCR) from genome of bovine TB. The PCR product of ESAT-6 was cloned into expression vector pET22b to reconstruct the pET22b-ESAT-6 recombinant plasmid. The PCR product of MPT83 was cloned into expression vector pET28a and to reconstruct the pET28a-MPT83 recombinant plasmid. The recombinant plasmids were transformed into E.coli DH5a arid BL21 respectively, and the positive recombinant was identified by the enzyme, PCR and sequenceing. Recombinant proteins ESAT-6 and MPT83 were expressed after inducted by IPTG and analyzed by SDS-PAGE and Western blotting. The immunogenicity of the expressed proteins was identified by Western blotting by using the bovine serum positive for anti-TB antibodies. The results are very important for the study on ELISA detection and vaccine of Bovine TB.Then the recombinant proteins ESAT-6 and MPT83 were purified with His-Bind affinity chromatography. The inclusion bodies were fully renaturated by semipermeable membrane. Development of indirect ELISA method based on the two recombined proteins for bovine TB detection. Based on previous studies, an indirect ELISA with ESAT-6 and MPT83 as coating antigens were improved in many aspects including coating antigen concentration, serum concentration, HRP concentration, incubation time for enzyme-substrate reaction and the method of result interpretation. Test of 285 samples from bovine with positive skin test of PPD,74 samples from bovine with negative bovine skin test of PPD and 79 samples from bovine with positive test of IFN-y showed a coincidence rate of 62.1%,98.64% and 83.54% of ESAT-6-ELISA and a coincidence rate of 57.19%,100% and 78.48% of MPT83-ELISA against cattle serum,respectively.The specific antigens-based bovine TB ELISAs were established, which can be used in extensive survey and monitoring of bovine tuberculosis in farms.In summary, we have expressed secreted ESAT-6 and MPT83 proteins of bovine TB by molecular technique. The specific antigens-based bovine TB ELISAs were established by using ESAT-6 and MPT83 proteins, they have potential to be further developed to a bovine TB diagnostic kit, which will provide technical support for the prevention and control of bovine TB in china.