Cloning, Expressing, Purifying Secreted Protein Ag85B Of Mycobacterium Tuberculosis And Its Immunological Properities Study

Tuberculosis(TB) is a chronic respiratory infectious disease caused by thepathogen Mycobacterium tuberculosis. TB remains an important global public health problem, because about one-third of the world's populations are infected with the M. tuberculosis and there are 10 million new cases and 3 million deaths annually. In recent years, such factors as increasing frequency of drug-resistance M.tuberculosis isolates and incidence of HIV-associated tuberculosis and increasement of population movement, have aggravated further the opportunity of people worldwide face to the threat of TB.It has been proved that some secreted protein of MTB could induced cell-mediated immune responses in many animal models. Among these, several antigens have showed higher protection efficacy in animal models such as Ag85 complex. ESAT6, MPT64, Hsp65 and PstS3. etc.Sinha et al compared Immunobiological properties of six different secreted protein and found that 30 kDa secretory protein of M. tuberculosis is the key immunoprotective antigen and may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.In this paper,we constructed the plasmid pPRO EX HTb'TB3?which contains the Ag85B gene, then we transformed the PPRO Ex HTb-TB3?into E.coli DH5 a andexpressed it high-efficiently.Immunized the BALB/c mice with Ag85B,BCG and saline respectively,after immnization,expression of Ag85B in vivo was assessed indirectly by the production of anti-Ag85B antibodies, Ag85B and BCG could induce high liter specfic antibody against Ag85B in immunized mice. To evaluate cell-mediated immune response, level of IFN y secreted by splenocytes of immunized mice which upon antigen-specific stimulation was detected by using a cytopathic effect reduction assay and splenocytes proliferation of immunized mice in response to antigen restimulation was measured by MTT method. Significant increased levels of IFN V could be measured in spleen cell culture supernatants from BALB/c mice immunized with Ag85B and BCG. IFN y levels in culture supernatants of spleen cells from BALB/c mice immunized with Ag85B and BCG were 960pg/ml and 814pg/ml, respectively. But IFN Y levels in that of saline immunized was below 80pg/ml. Lymphoproliferation in response to antigen restimulation in vitro was measured. BALB/c mice immunized with Ag85B and BCG demonstrated significant lymphocyte proliferation responses as seen by the elevated stimulation indexes, which both are twofold more than that of control mice. Spleen cells from negative control mice did not proliferate upon restimulation (both stimulation indexes under 1.5).In summary,immunized with Ag85B expressed by prokaryotic expression plasmid could induce humoral immunity responses and protective cell-mediated immuniry.lt suggested that Ag85B may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.