Mycobacterium Bovis Ag85A-MPB70-MPB63-MPB64Fusion Protein In The Diagnosis Of Bovine Tuberculosis ELISA

Bovine tuberculosis is caused by Mycobacterium bovis, a human and animal co-infected with chronic wasting diseases. Bovine tuberculosis pathogen can infect a variety of animals, and cattle are most susceptible animals, especially cows. According to the survey of more than10%of human tuberculosis caused by M. bovis. Getting closer and closer the relationship between humans and cattle, M. bovis is an important pathogen of human tuberculosis, causing a huge threat to public health and safety, mastery and application of highly sensitive, highly accurate diagnosis of bovine tuberculosis is effective in preventing human be an important means of infection by M. bovis.In this experiment, the recombinant plasmid the pMD-18-T-63-64and ag85a, mpb70PCR recovery product as a template by PCR amplification of Mycobacterium bovis gene ag85a, mpb70, and mpb63-64. By overlapping gene fragments Ag85A and mpb70as a template, an extension of the shear technology amplified fusion gene ag85a-70, and then to ag85a-70and mpb63-64as a template, using the SOE PCR technique successfully fusion gene ag85a-70-63-64.The prokaryotic expression vector the pET-30a (+) on the fusion gene ag85a-70-63-64clone by sequencing and by Nco Ⅰ, EcoR Ⅰ double digestion identification, to successfully build a positive recombinant plasmid pET-30a-ag85a-70-63-64. Positive recombinant plasmid pET-30a-ag85a-70-63-64was transformed into feelings of competent cells E. coli BL21(DE3) and induced by IPTG after SDS-PAGE and Western blotting analysis, the results show the successful expression of the target protein.and positive serum reaction with bovine tuberculosis, M. bovis antigen reactivity. By cellulose ion exchange chromatography on DEAE-52purified fusion protein of high purity Ag85A-MPB70-MPB63-MPB64.At the same time this experiment, the purified fusion protein Ag85A-MPB70-MPB63-MPB64as a diagnostic antigen, its package of liquid concentration, serum dilution, the closure time of the role of fluid and substrate selection and optimization, the initial est ablishment ofbovine tuberculosis an indirect ELISA. The test confirmed the fusion protei n of Ag85A-MPB70-MPB63-MPB64make the diagnosis of bovine tuberculosis antigen has a good specificity and sensitivity.