Production And Antigenicity Analysis Of Specific Antigen Of Mycobacterium Tuberculosis

It is well known that tuberculosis is caused by Mycobacterium tuberculosis infection. Mycobacteria tuberculosis proliferate and release plenty of antigens especially protein antigens in the body, and antigens selectively stimulate B cell to produce Immunoglobulin. At the early stage of acute infection, IgA and IgM take the predominance, then IgG appears, the level of IgG is associated with the state of infection. So detection of antibody to anti-TB is accessary for the diagnosis of tuberculosis. For there is a panel of mycobacterial antigens to induce immune response, the purpose of this research is to find higher sensitivity and specificity antigens by recombining and expressing protein antigens of Mycobacterium tuberculosis, and by isolateing proteins and lipoglycans antigens from Mycobacterium tuberculosis and analysing their antigenicity.Firstly, to extract DNA from standard strain of H37Rv as the template, design primers according to the sequence of gene of protein from GeneBank,amplify gene of 38KDa and 14KDa by PCR, insert to PET-30a(＋)and PET-22b(＋) and express proteins respectively in inclusion body and solubility, purify by Nickel affinity chromatography,at last get target proteins of higher purity, and Western blot assays showed that two recombinant proteins have satisfactory antigenicity .Secondly, to attain cellular proteins and lipoglycans of Mycobacterium tuberculosis, standard strain of H37Rv was grown on Sauton liquid medium for 3~4 weeks, the bacterial cells heat-killed were collected and disintegrated in ice by sonication, cellular proteins were extracted with phenol/chloroform; the water-soluble fraction was treated with chloroform/methanol,DNase I and RNase, a step of dialysis removed lipide, residual phenol and nucleotides from the glycans and lipoglycans(LPS-1); Triton-X114 phase separation technique was applied on LPS-1, Triton-X114 formed a clear micellar solution at 4℃ and two phases at 37℃, a hydrophilic detergent- depleted phase (LPS-2)containing AMS and an amphipathic detergent- rich phase (LPS-3)containing LAMS and LMS.Lastly, to coat five kinds of protein antigens including PPD,recombinant 38KDa,14KDa,ESAT-6,cellular proteins and three kinds of LPS antigens, use HRP-goat-anti-human-IgG as the second anbody, detect TB postive and negative refference serum based on the mechanism of indirect ELISA. Results showed that the sensitivity of the four kinds of protein antigens including PPD, 38KDa,14KDa,ESAT-6 and LPS-1 were 87.8％,80.5％,75.6％,96.0％,71.7％ respectively, and the specificity of the other four antigens except ESAT-6 would reach to 100% if ignoring NO.24 negative serum which showed a postive response. All results showed that those five antigens have good antigenicity, but the sensitivity of cellular proteins of Mycobacterium tuberculosis and LPS-3 were only 24.4％and 43.9％, which was lower than reports. The sensitivity of LPS-2 was consistent with reports. The research selected five kinds of antigens of Mycobacterium tuberculosis which have satisfactory antigenicity from eight kinds , providing basic study for production of anti-TB diagnostic kits.