Studies On Construction And Immune Mechanism Of Recombinant Bb-ESAT-6-MPT64 Vaccine Of Mycobacterium Tuberculosis

ObjectiveTo construct the recombinant Bb-ESAT-6-MPT64 vaccine with Mycobacterium tuberculosis ESAT-6 and MPT64 antigen coding gene;To analyse the expression of Escherichia.coli-Bifidobacterium shuttle plasmid pGEX-ESAT-6-MPT64 induced by IPTG in BL21(DE3);To detect the levels of serum IgE,IgG and its subclasses,the proliferation of splenocytes,the levels of CD4+ and CD8+ subsets of T lymphocyte,the apoptotic rate of splenocytes after immunization with vaccine by different inoculation routines against subsequent challenge with H37Ra.If so,the protective immunity induced in BALB/c mice of the recombinant Bb-ESAT-6-MPT64 vaccine could be explained and a kind of valuable vaccine to control tuberculosis would be provided.MethodsThe ESAT-6 and MPT64 genes were amplified by PCR from MTB H37Rv.By Gene SOEing technique,a fusion gene was constructed by splicing ESAT-6 gene and MPT64 gene with (Gly4Ser)3,then cloned into Escherichia coli-Bifidobacterium shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-ESAT-6-MPT64.The recombinant plasmid was introduced into BL21(DE3) and Bb by electroporation to construct recombinant vaccine Bb-ESAT-6-MPT64.The target protein induced by IPTG in BL21(DE3) for 1~13h was analysed by SDS-PAGE and Western blot.In order to study protective immunity induced in BALB/c mice by immunization with the recombinant Bb vaccine against subsequent challenge with MTB. 65 female BALB/c mice were randomly divided into 5 groups,13 in each group.Group A:rBb-ESAT-6-MPT64 was subcutaneously injected;Group B:rBb-ESAT-6-MPT64 was intramuscularly injected.Group C:rBb-ESAT-6-MPT64 was intranasally injected;Group D:Bb was subcutaneously injected;Group E:100μl PBS served subcutaneously as control.5×106CFU vaccines were suspended in 100μl PBS and subcutaneously injected once for group A and intramuscularly injected for group B.5×105CFU vaccines were suspended in 10μl PBS and intranasally inoculated once for group C.All the mice were intranasally challenged with 5×105CFU H37Ra of Mycobacterium tuberculosis on 8w after immunization. After 6w, serum IgG,IgG1,IgG3,IgG2a,IgG2b and IgE were respectively detected by ELISA, the proliferation of splenocytes were measured by MTT method, the number of CD4+ and CD8+ subsets of T lymphocyte were detected by FCM and the apoptotic rate of splenocytes with or without ConA stimulation were examined by Annexin V-FITC kits. ResultsESAT-6-MPT64 fusion gene identified by Agarose gel electrophoresis was about 1020bp,which was in accordance with the expected result by sequence analyzing.Restriction-endonuclease digestion demonstrated that ESAT-6-MPT64 fusion gene was correctly cloned into the multiple cloning site of Escherichia coli-Bifidobacterium expression shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-ESAT-6-MPT64. It was confirmed that the recombinant plasmid was successfully introduced into Bb by analyzing the rBb selected in MRS medium with 50μg/ml Amp by PCR.SDS-PAGE and Western blot showed the recombinant plasmid was able to express target protein in E.coil BL21(DE3) inducing by IPTG for 1~13h,which was expressed as a band of 60kDa.The content contained 22% of total bacterial protein of E.coil and could combine with specific antibody in mice serum.On the 6th week after mice challenged with H37Ra, the levels of serum specific antibodies were detected by ELISA:for groups A~C,the levels of antibodies IgG,IgG1,IgG2a,IgG2b were remarkably higher than control groups D and E; the levels of IgG,IgG1,IgG2a,IgG2b for group C were remarkably higher than group A and B,but there were no differences among all groups for IgG3 and IgE ;All antibodies for group A had no difference with group B.The proliferation of splenocytes were measured by MTT method: for groups A~C,the stimulating index(SI) of the original groups,the groups with MTB and ConA stimulation were remarkably higher than the control groups D and E,each group with ConA stimulation was remarkably higher than the corresponding original group or the MTB stimulation one;each group with MTB stimulation was remarkably higher than the corresponding original one.In the original groups,there were no differences among group A,B and C.In the groups with MTB stimulation,the SI of group C was remarkably higher than group B. In the groups with ConA stimulation,the SI of group C was remarkably higher than both group A and B. There was no difference between group A and B.The levels of CD4+ and CD8+ subsets of T lymphocyte were detected by FCM: for groups A~C,the levels of CD8+ subsets were slightly higher than the control groups D and E,but had no statistic value.the levels of CD4+ subsets were remarkably higher than both control groups,which for group C were remarkably higher than group A and B.The apoptosis of splenocytes were detected by Annexin-V method. for groups A~C,the apoptotic rate of the original groups and the groups with ConA stimulation were remarkably lower than the control groups D and E,each group with ConA stimulation was remarkably higher than the corresponding original group; the apoptotic rate for group C were remarkably lower than group A and B,which for group A were remarkably lower than group B.Conclusion1.The ESAT-6-MPT64 fusion gene was successfully amplified by Gene SOEing technique.2.The shuttle expression plasmid pGEX-ESAT-6-MPT64 was successfully constructed.3.The recombinant vaccine Bb-ESAT-6-MPT64 was successfully constructed.4.The recombinant plasmid pGEX-ESAT-6-MPT64 was able to express 22% target protein in E.coli BL21(DE3) induced by IPTG,which was of specific antigenicity.5.The recombinant Bb-ESAT-6-MPT64 vaccine was able to elicit strong specific CD4+Th1 cellular immune,humoral immune and weak CD8+CTL respones.6.The protective immunity induced by the recombinant Bb-ESAT-6-MPT64 vaccine with intranasal inoculation is better than that of subcutaneous or intramuscular injection.