| Objective: In order to construct and prepare a novel fusion protein vaccine containing Mycobacterium tuberculosis(M. tuberculosis)latent antigen, this study was designed to screen M. tuberculosis antigens with higher immunogenicity in latent infection. With the combination of antigens from dormant stage and growth stage of M. tuberculosis, it does good to develop novel TB subunit vaccine which is more protective. Furthermore, constructing a pulmonary latent BCG infection mouse model and using this model to preliminarily evaluate the therapeutic effect of M. tuberculosis subunit vaccine.Methods: 1).With the help of bioinformatics technology, we got 5 genes(Hsp X, Rv1738, Rv2626 c, Rv2007 c and Rv3130c)related to TB latent infection. These antigens were respectively cloned and expressed in E. coli. Based on the different immune responses in TB patients, latent TB patients and healthy people the immunigenicity of these antigens were evaluated, and the antigens with highest immunogenicity were screened. 2).Constructing the fusion protein LT40( p ET30a-ESAT6-Rv1738-Rv2626c) and LT28(p ET30a-CFP10-Hsp X) by genetic engineering technology and expressing and obtaining the fusion protein LT40 in E. coli. 3). To analyze the immunogenicity of LT40 on mice model. The LT40, ESAT6, Rv1738 and Rv2626 c proteins were respectively injected into mice at 0-3-6 week. The mice were sacrificed to detect the antibody level in serum and IFN-γ secretion level in the spleen cells at 12 th week. 4).The mice were infected with BCG(5×106CFU)by intratracheal administration. Ater BCG infection, the mice were treated with dexamethasone by intramuscular injection. Then, the mice were sacrificed to obtain lung at 2day, 4week, 8week and 12 week. At last, the lungs were used for determination of bacillary load. The model was constructed successfully or not depending on the detection of the above figure. That’s to say, when we didn’t detect any pulmonary bacterial on lung tissue after BCG infection, but large numbers of pulmonary mycobacteria emerged after the dexamethasone treated, this model was successfully constructed.5).The therapeutical effect of the fusion protein vaccines LT40 and LT70(ESAT6-Ag85B-MPT64<190-198>-Mtb8.4-Rv2626c) were evaluated by the mice model we constructed. Our laboratory has confirmed that LT70 can protect mice against BCG attack. After infected with BCG, mice were assigned to five groups: 1. PBS group; 2. adjuvant group; 3. LT40 group; 4. LT70 group; 5. LT70+LT40group. Group 3 to 5 were immunized with fusion protein vaccines by inguinal subcutaneous injection method at 12, 14, 16 weeks, the PBS and adjuvant group were treated with PBS and adjuvant as control. All of the mice were injected with dexamethasone by intramuscular injection 3 times(once every week). At week 25, the mice were executed with cervical dislocation methods, the lungs were taken out for CFU count.Result: 1. Among five antigenss Hsp X, Rv2626 c, Rv2007c(Fdx A), Rv1738 and Rv3130 c, which related to M. tuberuclosis latent infection, Rv1738, Rv2626 c, Rv3130 c, Hsp X antigens induced higher Ig G antibody level and IFN-γ levels in TB close contacts comparing with TB patients and healthy donor. But in TB patients, antigen ESAT6 and CFP10 can induce higher IFN-γ levels than that in healthy donor. We predict that Rv1738, Rv2626 c and Rv3130 c play an important protective effect in the process of protecting body from TB infection. Among them, Rv1738 has the highest immunogenicity in cellular immunity and humoral immunity. Therefore, Rv1738 is one of most important protective antigen among them in M. tuberculosis latent infection. 2. Linking the M. tuberculosis growing antigen ESAT6 and latent antigen Rv1738 and Rv2626 c to construct the fusion protein LT40, which was expressed stably in the E. coli in the form of inclusion and obtained massive and higher purity of the protein. Unfortunately, the fusion protein LT28, based on the growing antigen CFP10 and latent antigen Hsp X, expressed poorly, and the protein was not obtained. 3. The fusion protein LT40 can induce higher level of antibody(Ig G1 and Ig G2c)in serum and IFN-γ level in spleen lymphocytes against LT40 antigen stimulation in LT40 immunized mice, but LT40 has a weak immune reaction with the stimulation of single antigens. It indicated that the immune response induced by fusion protein LT40 is different from natural antigen. 4. We constructed a pulmonary latent BCG infection mouse model successfully. Mice were infected with BCG by nasal drip. We didn’t detect any pulmonary bacterial on lung tissue 12 weeks later. But after the intervention of dexamethasone, plenty of pulmonary bacterial reemerged from the lung tissue. 5. Using the established pulmonary BCG latent infection mouse model to evaluate the therapeutic effect of protein vaccine. The BCG colony forming units results showed that: PBSgroup showed higher CFU counts than other groups, which has a lot of mycobacteria outbreak after immune suppression; Compared with PBS group, vaccine group and adjuvant group showed significant lower CFU counts, and vaccine group(LT40 group and the LT70 group and LT40+LT70 group)showed the lowest CFU counts. It showed that protein vaccines LT40 and LT70 had some ability to clear the latent infected BCG.Conclusions: 1. The latent TB antigen Rv1738 has the highest immunogenicity in cellular immunity and humoral immunity among antigens(Hsp X, Rv2626 c, Rv2007 c, Rv1738 and Rv3130c), and Rv1738 is one of protective antigen in MTB latent infection. 2. The fusion protein LT40 was successfully constructed, expressed and obtained stably in the E. coli in the form of inclusion. The fusion protein LT40 can induce higher level of antibody(Ig G1 and Ig G2c)in serum and IFN-γ level in spleen lymphocytes against LT40 antigen stimulation in LT40 immunized mice, but LT40 has a weak immune reaction with the stimulation of single antigens. It indicated that the immune response induced by fusion protein LT40 is different from natural antigen. 3. The pulmonary latent BCG infection mouse model was successfully constructed, which provided a simple animal model to preliminarily evaluate the therapeutic effect of TB subunit vaccine. 4. The protein vaccines LT40 and LT70 have some ability to clear the latent infected BCG. |
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