Expression, Purification Heat Shock Protein 16.3 Of Mycobacterium Tuberculosis And Its Immunological Properties Study

Tuberculosis (TB) is a chronic infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). Now, one-third people are infected worldwidely by MTB, in which ten percent are able to become active disease and the others stepped in latent tuberculosis infection(LTBI). The LTBI is a subclinical state when the MTB is dormant, which infected so many, extensive and difficultly diagnosed persons, making it as the main cause of the TB happence currently.Heat shock protein 16.3(Hsp16.3) is expressed by hspX (Rv2031c, acr) gene in Mycobacterium tuberculosis. Many reports said this protein was closely related to MTB dormancy. Some studies have confirmed that Hsp16.3 was a target antigen against TB, for possessing T-cell epitopes of human and mouse. In this study, we compared the immune responses and protection ability against MTB infection of Hsp16.3 expressed and purified to the synthetic peptide of Hsp16.3 with different adjuvants(DDA+MPL,FIA), intending to learn the impaction of adjuvant choosing on immune responses, and learn more about the immunological properties of Hsp16.3 and synthetic peptide in order to accumulate experiment datum for exploiting new vaccines for TB and settle physical base for researching the mechanism of MTB entering and survival in macrophage under dormant condition. It's meaningful to control and eradicate TB that methods for inhibiting dormancy forming and killing the dormant bacteria were found out.1. Expression and purification of Hsp16.3hspX gene was amplified by polymerase chain reaction(PCR)from genome DNA of MTB H37Rv strain, and inserted into cloning vector pTA2 for sequencing purpose. The genetic sequence of hspX were identical with that of Genbank reported, then digested by restriction endonuclease and cloned into expression vector pPROEX HTb. The expressed protein was confirmed by western blot analysis with mouse-specific monoclonal antibody against 6×His and 16 kDa protein with a bind at 16 kDa respectively. Then, it was purified by Ni-NTA purification system under denaturing condition as description in the protocol.2. Immunological properties research of Hsp16.3 and its synthetic peptide Three trail groups were Hsp16.3+ dimethyl dioctadecylammonium bromide (DDA)+ Monophosphoryl Lipid A (MPL), synthetic peptide+DDA+MPL and synthetic peptide+FIA, while two control groups were BCG group and saline group(ten in per group). BALB/c mice were inoculated subcutaneously three times at 2 weeks interval. And the antibody-specific concentrate in sera of the immunized mice were tested at 0,2,4,6,8 weeks during the immunization with Hsp16.3 and synthetic peptide coating respectively by ELISA method. The antibody titers of the sera obtained at 8th week was also measured by ELISA with the same antigens coating. We could observe that antibody-specific to Hsp16.3 and the peptide increased rapidly in the former 4 weeks and moderately in the later weeks. The average antibody-specific titers to Hsp16.3 of Hsp16.3 protien+DDA+MPL, synthetic peptide+DDA+MPL, synthetic peptide+FIA, BCG and saline were 1:3000,1:1500,1:1500,1:500 and 1:100 respectively . While, the average antibody-specific titer to synthetic peptide for the five groups were 1:1500,1:1500,1:1000,1:500 and 1:100. Obviously, BCG group was much lower than three trail groups (P <0.01), with significant difference compared to saline group(P <0.05).Half immunized mice in each group were sacrificed for single splenocyte four weeks after the final immunization. The index of splenocytes proliferation (SI) in response to Hsp16.3 and synthetic peptide was measured by MTT assay. The levels of IFN-γwere detected by indirect ELISA with the same two antigens stimulation. The data showed SI of Hsp16.3+DDA+MPL, synthetic peptide+DDA+MPL and synthetic peptide+FIA were significantly higher than BCG group(P <0.05) and saline group(P <0.01). With the Hsp16.3 stimulating, the SI of Hsp16.3+DDA+MPL and synthetic peptide+DDA+MPL were a little higher than synthetic peptide+FIA(P <0.05), as well as with the synthetic peptide stimulating, the SI of synthetic peptide+DDA+MPL was higher than Hsp16.3+DDA+MPL (P <0.05).The levels of IFN-γof Hsp16.3+DDA+MPL, synthetic peptide+DDA+MPL and synthetic peptide+FIA were lower than BCG group(P <0.01) and higher than saline group (P <0.01). With the Hsp16.3 stimulating, Hsp16.3+DDA+MPL, synthetic peptide+DDA+MPL and synthetic peptide+FIA didn't display remarkably different, as to with the synthetic peptide stimulating, synthetic peptide+DDA+MPL was significantly higher, compared to Hsp16.3+DDA+MPL and synthetic peptide+FIA (P <0.05).The other half mice were intravenously attacked by with MTB H37Rv. Immunized mice were sacrificed at fourth week post-infection. Homogenized spleen tissue was serially diluted and placed on 7H10 at 37℃. The cloning forming units(CFUs) of MTB in spleen were calculated three or four weeks later. The outcomes showed compared to saline group, other four groups all had much better resistance to MTB H37Rv(P <0.05), but compared to BCG group, the resistances of Hsp16.3+DDA+MPL, synthetic peptide+DDA+MPL and synthetic peptide+FIA were all subordinate to BCG(P >0.05).3. ConclusionHsp16.3 was purified successfully in E.coli DH5α. The results of immunization indicated that the DDA and MPL mixture as adjuvant was better than FIA in this study, and showed that both Hsp16.3 and synthetic peptide induced cell-mediated immune responses and humoral-mediated immune responses and produced effective resistance to MTB H37Rv successfully. Hsp16.3 induced higher specific antibody, but lower SI, IFN-γreleasing level and resistance against H37Rv compared to synthetic peptide with no statistics significance. Hsp16.3 and synthetic peptide have some common immunological characteristics, while they have their own merits. In conclution, both of them deserve the candidates for TB vaccine.