The Prokaryotic Expression Of Mycobacterium Bovis Gene Mpb51-63-83-85b And Application Of The Product

Bovine TB(Bovine tuberculosis),caused by Mycobacterium bovis (Mycobacterium bovis),is a zoonotic chronic and expendable disease. and it was classified as the second rank disease in our country. In recent years, this disease is not only has been effectively controlled, but also the prevalence is rising, and influence the development of stock raining severely and menace the health of human beings. MPB51, MPB63, MPB83 and Ag85B of Mycobacterium tuberculosis are the major protective antigen. In serological diagnosis of Bovin tuberculosis has great potential,as bovine tuberculosis of subunit vaccine and DNA vaccine a good candidate antigen against bovine tuberculosis. 1. Obtain the fusion gene mpb51-63-83-85b of M.bovisThe DNA frgments of mpb51-63,mpb83 and ag85b were amplified separately by PCR from pMD-18-T-51-63 and pGEM-T-83, pGEM-T-85b, DNA frgments of mpb83-85b obtained from mpb83 and ag85b by SOE (splicing by overlapping extension).Then obtained fusion gene mpb51-63-83-85b from DNA frgments mpb51-63 and mpb83-85b by SOE PCR (splicing by overlapping extension polymerase chain reaction, SOE PCR). 2. Expression of fusion gene mpb51-63-83-85b of M. bovisThe DNA frgments of mpb51-63-83-85b and pET30a(+)were digestde by Nco I and EcoR I double enzymes, recycling and purification the fragments, and use the T4 DNA ligase, successfully constructed the prokaryotic expression vector pET30a-51-63-83-85B. Plasmid containing pET30a-51-63-83-85B was transformed into competenceE.coliBL21(DE3), The bacterium was induced by IPTG and its lysate was loaded directly onto SDS-PAGE.An approximately 100kDa protein was observed on the SDS-PAGE.The protein of MPB51-MPB63-MPB83-Ag85B was analyzed by using Western-blotting and it had antigenic activity of MB. Through the fusion protein was purified by ion exchange chromatography. These results provide the basis for the development of subunit fusion gene mpb51-63-83-85b and its expression product as a DNA vaccine, subunit vaccines and new diagnostic antigen and the establishment of bovine tuberculosis specific and sensitive diagnostic method for the foundatio 3. Application of the MPB51-MPB63-MPB83-Ag85B to M. bovis in ELISA detection of bovin tuberculosis.The fusion protein of MPB51-MPB63-MPB83-Ag85B purified as a diagnostic antigen, The serum samples from 90 healthe ows were tested, as the the cutoff value to establish an indirect iELISA method. Passed detection positive serum, negative serum and paratuberculosis-positive serum, the results show that the method has good sensitivity and specificity. Taken PPD as the reference text,283 serum sample were assayed with iELISA.As a rusult, the fusion protein MPB51-MPB63-MPB83-Ag85B positive detection rate of 5.30%(15/283), PPD positive detection rate of 6.36%(18/283), both the positive coincidence rate was 83.33%. Preliminary results show that the fusion protein MPB51-MPB63-MPB83-Ag85B as the diagnosis of bovine tuberculosis ELISA of antigen has good sensitivity and specificity.