| Bovine tuberculosis (TB) is caused by the Mycobacterium bovis. Both animal species and human beings can be infected. It was classified as the second rank disease in our country and must be immuned by force. In recent years, the development of stock farming and the health of human beings were still threatened by TB. According to the report from the World Health Organization (WHO), one third of the world population was in the latent period of TB. The number of the patient is around20billion now, and9billion new influents were occured each year with2billion deaths. It was more serious than the total number of AIDS, malaria and diarrhoea. There were an estimate1.3million incident cases in our country. China is one of the22high-burden countrys, which account for approximately80%of all new TB cases arising each yeat. There was amount3million persons fall in TB by the infection of Mycobacterium bovis.The clinical feature of TB was not typical. Lots of diagnostic method was used domestic and overseas with their own merit and demerit. Bacteriology diagnosis was simple to use, but the positive was low. The molecular biology diagnosis required precise instrument and complicate technology, and it used in lab most of the time. Immunology diagnosis had low specificity, which could be increased by unite of several methods. At present, the PPD allergic reaction had been identified as the only method for TB diagnosis by OIE. But some defects still existed. First, its low specificity caused indistinguish of different kinds of mycobacteria. It was limited to test lived animal samples and not suitable for TB census to the wild animals. Also, it cannot figer out the cow infected by BCG immunity or natural infection. IFN-y rest had been identified as the latest diagnosis method to TB in Australian by virtual of its higher specificity and sensitivity. But its limitation was the high cost and antigenic cross-reaction. Due to the merit of easy to operate and did not need precise instruments, ELISA diagnosis was widely used in serolofy aspect. Its limitation was specificity and sensitivity. BCG, as the most effective vaccine in the prevention of TB, had postponed the prevalence of the disease. However, with many defects, BCG brings lots of difficulties to the quarantine. For example, the immune effect after immunization in different zones and organisms was instability. So, the study of newly prawparatum toTB prevention and diagnosis became more important.In order to prevent and detect TB more efficient and to cut down the morbidity of TB in human beings, in this study, the ag85a and mpb63were amplified by PCR, from the recombinant cloning plasmid pGEM-T-ag85a and pGEM-T-mpb63respectively, and two fragment with770bp and500bp were obtained. The fusion gene ag85a-mpb63was constructed by SOE-PCR. It was cloned into pMD-18-T vector, and the recombinant plasmid pMD-85a-63was gained. The sequence analysis showed that the fusion gene fragment ag85a-mpb63was1320bp, and it had100%homology in nucleotide sequence respectively compared with the ideal one. The fusion gene ag85a-mpb63was subcloned into the expression vector pET-28a (+), and the recombinant plasmid pET28a-85a-63was gained. The recombinant plasmid expressed a50kDa fusion protein in the E.coli BL21(DE3) that induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Analyzed by western-bolt, the fusion protein was of antigenic reactivity of Mycobacterium bovis. Broken by the ultrasonic and analyzed by SDS-PAGE, the fution protein mainly existed in the cell sedimentation. Pure protein was got by ion exchange chromatography. The purified protein used as a diagnostic antigen, an indirect ELISA method was founded.282tested sera were diagnosed by the purified protein using the method of iELISA, the positive rate by Ag85A-MPB63is5.67%while is6.73%by PPD, and both positive rate is84.2%. It was showed that the fusion protein Ag85A-MPB63with a good specificity and sensitivity of the ELISA diagnosis antigen of bovine tuberculosis. |
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