Expression, Purification Secreted Protein MPT64 Of Mycobacterium Tuberculosis And Its Immunological Properities

Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). TB remains an important global public health problem, because about one-third of the world's populations are infected with the MTB and there are 8 million new cases and 3 million deaths annually. In recent years, factors such as the unsuccessful protection of BCG, increasing frequency of drug-resistance MTB, incidence of HIV-associated tuberculosis and increase mcnt of population movement, have aggravated further (he opportunity of people worldwide face to the threat of TB. There are difficulties in detecting new TB patient for the diagnosis reagent of TB by using purified protein derivative (PPD). PPD is not sensitive enough, furthermore BCG vaccination interferences the diagnosis of TB.For the reasons above, it is very important to develop new vaccine, or enhance BCG effection and diagnosis reagent for finding TB patient timely and specially.It has been proved that some secreted proteins of MTB could induced cell-mediated immune responses in many animal models, such as Ag85complex, ESAT-6, MPT64, and MPT32, etc. Among these, MPT64 is one of relatively important and specific antigens. It can induce delayed-type hypersensitivity in guinea pig, both nature and recombinated protein can induce protective T-cell responses, so it can be used both vaccine and diagnosis reagents of TB.In this paper, the gene encoding MPT64 protein was amplified by PCR from the genome of M. tuberculosis H37Rv strain. Using the prokaryotic expression vector pProEX HT, we successfully expressed the MPT64 protein in fusion form. There are two forms of MPT64 protein, one is soluble and the other is inclusion. The yield of expressing was analysised by the computer thin-layer scanning, and the proportion of this fusion protein was 13.5% of the whole proteins. We purified the fusion MPT64 protein by the Nickel-ion affinity chromatography.Then we tested the immunological characteristics of this fusion protein in mice. Immunized the BALB/c mice with MPT64 protein that was transferred to membrane beforehand. The method of subcutaneous embedding was used in the first two vaccinations; reinforcement vaccination was taken with the SDS-PAGE gel supernatant. After immnization, expression of MPT64 in vivo was assessed indirectly by the production of anti-MPT64 antibodies by HLISA, and the average titre of anti-MPT64 antibodies is 1:100.To evaluate cell-mediated immune response, level of IFNy 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 IFNy could be measured in spleen cell culture supernatants from BALB/c mice immunized with MPT64 and BCG. IFNy levels in culture supernatants of spleen cells from BALB/c mice immunized with MPT64 and BCG were 1024ng/L and 1470 ng/L respectively. But IFNy levels in that of saline immunized was below80ng/L. Lymphoproliferation in response to antigen restimulation in vitro was measured. BALB/c mice immunized with MPT64 and BCG demonstrated significant lymphocyte proliferation responses as seen by the elevated stimulation indexes, which are 2.3 and 3.1 respectively. Spleen cells from negative control mice did not proliferate upon restimulation. To further evaluate protective immune response to MTB, the vaccinated BALB/c mouse were infected with 5+105 CPU in tail vein, dramatic reductions compared to the saline injected mice were observed for spleen (0.9251ogio) CPU. The result that the protection of mice immunized with MPT64 are not good as that of BCG means that there is difference in the protection between the vaccine made of single antigen and BCG.In summary, we have expressed secreted MPT64 protein of MTB by molecular biology technique. Vaccination with recombinant MPT64 protein is an effective means to genenrate humoral immunity renpon...