High-level Expression Of Mycobacterium Tuberculosis PhoS2 Gene In E.Coli And Antigenicity Analysis Of The Recombinant Protein

Tuberculosis(TB) is one of the oldest infectious diseases for humans. Mankind has struggled with it for thousands of years, but it still is spreeding globally, threats seriously on human health and life. Every year, among all of the infectious diseases, the number of the patients died with TB is more than the number of the patients died with AIDS, malaria, diarrhea, tropical diseases together. It leads to the deaths of 300,000 children every year. One-third of the world's people (about 20 million) have been infected with Mycobacterium tuberculosis (MTB). In China, nearly half of the population (about 550 million) has been infected with Mycobacterium tuberculosis. Global TB epidemic has expanded rapidly, and it has posed a severe challenge on the international public health. When a person infected by the MTB, the Mycobacterium tuberculosis-specific antigen appeared firstly, so the detection of the antigen could be used as the direct evidence of infection with MTB. It could avoid the "false negative" caused by the low humoral immune and cell-mediated immunity. Therefore, the detection of the Mycobacterium tuberculosis- specific antigen has become a fast, specific, efficient and simple diagnostic method.In this study,PCR was used to amplify phoS2 gene fragment from Mycobacterium tuberculosis H37Rv genomic DNA firstly. Then, phoS2 was connected with the pGEM-T vector, identified by enzyme digestion and sequencing. Thirdly, through the enzyme digestion of the positive recombinant and the expression vector pET24b with Ndeâ… a nd Xhoâ… , phoS2 was connected with pET24b, and transformed into BL21. The expression products were identified by SDS-PAGE electrophoresis. The recombinant protein was purified by Ni-NTA affinity chromatography, and analysed by western blot and ELISA.Mycobacterium tuberculosis phoS2 gene was cloned, confirmed by enzyme digestion and DNA sequencing. Target gene was expressed efficiently in E.coli, the amount of the phoS2 protein in BL21 occupied more than 40% of total bacterial protein. Western Blot showed that the purified protein appeared strong reaction with serum samples of 6 TB patients, but had no reaction with 6 healthy controls. ELISA showed that the phoS2 gene's recombinant protein had a better diagnostic value of serology.In this study, it was found that the recombinant phoS2 protein had an excellent antigenic specificity and immunoreactivity, indicating a potential application value in diagnosis of tuberculosis. It made a foundation for further study of phoS2 gene in the serological diagnosis of tubeculosis.