Expression of Mycobacterium tuberculosis ferric uptake regulator A gene in Escherichia coli and generation of monoclonal antibodies to FurA.

Ferric uptake regulator A of Mycobacterium tuberculosis (MTB), which belongs to the Fur superfamily, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the pro-drug isoniazid. The feature and role of FurA in oxidative stress contribute to research on the pathogenesis of mycobacteria. In this study, four novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurA protein as immunogen. The furA gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pRSET-A and effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as soluble form in cell lysates and was purified via Ni-NTA purification system. Using the fusion protein to immunize BALB/c mice, four monoclonal antibodies (H9H6, H9E12, H10H6, and H10H8) were produced. As shown by Western blot analysis and cell fluorescence microscopy assay, the four antibodies could recognize the FurA protein, respectively. Then we assessed the effect of iron on the expression of FurA in MTB H37Rv and we concluded that iron does not affect FurA expression. These results suggest that the antibodies against FurA may provide a powerful tool for elucidating FurA biofunctions and regulation mechanism in the pathogenesis of tuberculosis.

[Immunoprophylaxis of recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis].

AIM: To evaluate immunoprophylaxis of recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis. METHODS: BALB/c mice were immunized with recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis. ELISA was used to detect the anti-MPT64 antibody titers and subtype in immunized mice sera. Splenocytes of immunized mice were separated and used to detect IFN-gamma and IL-12 levels, splenocytic proliferation, counts of CD4(+) and CD8(+) T cell percentage, cytolytic T lymphocyte specific lysis. The bacteria numbers in vaccination animal's lung and spleen were counted by colony forming units (CFUs) on plate. RESULTS: Recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis could induce high level of specific IgG antibodies, stronger T cell proliferation response, production of IFN-gamma and IL-12, CD4(+) and CD8(+) cell percentages and CTL effect in mice. And an efficacy protection against Mycobacterium tuberculosis was also observed in mice model. CONCLUSION: Recombinant Mycobacterium vaccae secreted MPT64 could induce high level humoral and cell mediated immune responses in mice and could be used as a candidate of new vaccine against TB.

[Immune response induced by Rpf domain from Micrococcus luteus in mice].

AIM: To investigate the immunobiology of Rpf domain from Micrococcus luteus. METHODS: BALB/c mice were immunized with Rpf domain three times at 2-week interval. ELISA was used to detect the title of the anti-Rpf domain antibody titer in the immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry. The levels of secreted IFN-gamma, IL-10 and IL-12 upon specific antigen stimulation was detected by ELISA. The Rpf domain immunized BALB/c mice were intravenously infected with 10(5) CFU MTB H37Rv. The number of CFU in the spleens was determined four weeks after final injection. RESULTS: The titer of the specific antibody in sera of the immunized BALB/c mice was 1:128 000. The SI of Rpf domain immunized group (2.10+/-0.12) was significantly higher than that of saline immunized group (0.90+/-0.21). The lever of IFN-gamma, IL-10 and IL-12 levels in culture supernatant of spleen lymphocytes from the fusion protein immunized mice was (1 126+/-36) ng/L, (368+/-13) ng/L and (289+/-14) ng/L, respectively, which was markedly higher than that of saline immunized group (P<0.01). Compared with normal saline immunized mice (6.64+/-0.13) four weeks after final injection, dramatic reduction in MTB replication was observed in the spleen (5.03+/-0.11) from the BALB/c mice immunized with fusion proteins. CONCLUSION: Rpf domain can be used as a candidate for a new TB vaccine.

[Preparation and characterization of monoclonal antibodies against Micrococcus luteus Rpf domain].

AIM: To express Micrococcus luteus Rpf domain in prokaryotic cells and prepare monoclonal antibodies against Rpf domain. METHODS: The gene encoding Micrococcus luteus Rpf domain was amplified from genome of Micrococcus luteus by polymerase chain reaction(PCR), and inserted into cloning vector pUC-19. After sequenced, Micrococcus luteus Rpf domain gene was subcloned into the expression vector pPro-EXHT and transfected into E.coli DH5alpha. After induced by IPTG, the bacteria controlled by T7 promoter expressed the fused Micrococcus luteus Rpf domain protein with a hexahistidine tail at its N-terminal and the target protein was purified under denaturing conditions. Using this protein as antigen to immunize the BALB/c mice and prepare monoclonal antibodies against Micrococcus luteus Rpf domain. Then specifities and relative affinities of mAbs were identified by ELISA. RESULTS: The fusion protein was purified by metal chelate affinity chromatography under denaturing condition. Three cloned mAbs were prepared from the mice immunized by Rpf domain. All of them could recognize Rpf domain. specifically. CONCLUSION: The prepared mAbs against Rpf domain have strong specificity with high titers, which provides useful tools for further study of the function of Rpf domain in TB prevention.

Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65 (Hsp65) with human interleukin-2 against Mycobacterium tuberculosis in BALB/c mice.

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin-2 (hIL-2) in BALB/c mice. We showed that the DNA vaccine pcDNA-Hsp65-hIL-2 could induce high levels of antigen-specific antibody, IFN-gamma, CD4(+) and CD8(+) T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.

[Immune response induced by Mycobacterium tuberculosis Rv2450 protein in mice].

AIM: To evaluate humoral and cellular immune response induced by Rv2450 protein. METHODS: Rv2450 protein was transferred to membrane and used to immunize C57BL/6 mice three times at 2 week interval. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry and the levels of secreted IFN-gamma, IL-10 and IL-12 upon antigen-specific stimulation was detected by ELISA. RESULTS: The titer of specific antibody in sera of the immunized C57BL/6 mice was 1:3 200. The SI of Rv2450 protein immunized group (3.76 +/- 0.19) was significantly higher than that of saline immunized group (0.89+/-0.17). The IFN-gamma, IL-10, IL-12 levels in culture supernatant of spleen lymphocytes from the immunized mice was (1 740 +/- 19) ng/L, (678 +/- 15) ng/L and (469 +/- 13) ng/L respectively, significantly different from that of saline-immunized group. CONCLUSION: Rv2450 protein can be used as a candidate for the new TB vaccine.

[Immune response and protective efficacy induced by fusion protein ESAT6-CFP10 of M.tuberculosis in mice].

AIM: To study murine humoral and cellular immune response induced by fusion protein ESAT6-CFP10 and to examine its protective efficacy against M. tuberculosis (MTB) in mice. METHODS: BALB/c mice were immunized subcutaneously on the back with fusion protein ESAT6-CFP10 that was transferred to nitro cellulose (NC) membrane beforehand. Stimulation index (SI) of the spleen lymphocytes of the immunized mice was measured by MTT colorimetry. The level of IFN-gamma and IL-2 and CTL upon antigen-specific stimulation were detected. The vaccinated BALB/c mice were intravenously infected with MTB H37Rv (10(5) CFU/mouse). Four weeks later the number of CFU in spleens was determined. RESULTS: The titer of serum specific antibody in BALB/c mice immunized with fusion protein ESAT6-CFP10 was 1:6,400. The SI of fusion protein immunized group (1.90+/-0.15) was significantly higher than that of saline-immunized group (0.9+/-0.15). The level of IFN-gamma and IL-2 induced by the fusion protein was 1.792+/-19 ng/L and 0.211+/-11 ng/L respectively, which was significantly higher than that of saline-immunized group and lower than that of BCG-immunized group. The specific killing activity of splenocytes was 36%. Compared with the saline-immunized mice (bacterial load was 6.51+/-0.13), MTB number (bacterial load was 5.24+/-0.15) was reduced dramatically in the spleens of BALB/c mice immunized with the fusion protein, but the protective efficacy of the mice immunized with BCG was higher than that of ESAT6-CFP10 vaccinated group. CONCLUSION: Fusion protein ESAT6-CFP10 can be used as a candidate for novel vaccines.

[Immunity characteristics induced by a genetic vaccine expressing Ag85B-ESAT6 fusion protein].

OBJECTIVE: To evaluate the humoral and cell-mediated immune responses induced by a genetic vaccine expressing the Ag85B-ESAT6 fusion protein, and to investigate its protective effect against Mycobacterium tuberculosis (MTB) challenge. METHODS: Fifty BALB/c mice were randomized into 5 groups and subjected to the following treatments respectively: immunization with normal saline, BCG, pcDNA3, A(Z)-pcDNA3-E(F) and E(Z)-pcDNA3-A(F) for 3 times at 2-week intervals. The stimulation index (SI) of the splenic lymphocytes from the immunized mice was measured by the methyl thiazolyl tetrazolium (MTT) method, and the level of secreted IFN-gamma upon antigen-specific stimulation was detected by ELISA. The immunized mice were intravenously infected with 10(5) colony forming unit (CFU) of MTB H(37)Rv. The numbers of MTB CFU in spleens were determined 4 weeks later. RESULTS: The specific antibody titers in the sera of mice immunized with plasmid A(Z)-pcDNA3-E(F) and E(Z)-pcDNA3-A(F) were 1:1,000 and 1:1,500 respectively, and the SI was 2.2 and 2.4 respectively, while the SI of the normal saline group and the plasmid pcDNA3 immunized group was only 0.9 and 1.1 respectively. The IFN-gamma concentrations in cultured supernatant of splenic lymphocytes from mice immunized with plasmid A(Z)-pcDNA3-E(F) [(5.48 +/- 0.38) ng/ml] and E(Z)-pcDNA3-A(F) [(5.76 +/- 0.51) ng/ml] were significantly higher than those of the normal saline group [(0.50 +/- 0.25) ng/ml] and the plasmid pcDNA3 immunized group [(1.20 +/- 0.33) ng/ml, P < 0.05], but were not significantly different with that of the BCG immunized group [(5.55 +/- 0.31) ng/ml]. Compared with plasmid pcDNA3 immunized group, the bacterial load (lg, CFU/g) in spleen was 6.08 +/- 0.25 which dramatically reduced in mice immunized with recombinant plasmids, but the protective efficacy of mice immunized with plasmid A(Z)-pcDNA3-E(F) (4.63 +/- 0.11) or E(Z)-pcDNA3-A(F) (4.50 +/- 0.32) was lower than that of the BCG vaccination group (4.09 +/- 0.27). CONCLUSION: The cell-mediated immune response induced by genetic vaccine expressing the Ag85B-ESAT6 fusion protein was similar to that induced by BCG immunization.

[Screening and construction of recombinant BCG strains expressing the Ag85B-ESAT6 fusion protein].

OBJECTIVE: To screen and construct recombinant BCG strains which express the Ag85B-ESAT6 fusion protein. METHODS: The heat shock protein 60 (Hsp60) and the alpha-ss signal peptide encoding sequence were amplified by PCR from Mycobacterium tuberculosis H(37)Rv and cloned into E. coli/Mycobacteria shuttle vector-pOLYG. The resulting expression vector was named pDE22, and then ag85b and esat6 genes were cloned into pDE22 at different sites. The resulting recombinant plasmids Ag85B-ESAT6 and ESAT6-Ag85 were electroporated into BCG. Positive clones were screened by hygromycin resistance and confirmed by PCR. Recombinant BCG culture supernatants were collected and analyzed by SDS-PAGE and Western blot. RESULTS: Two recombinant BCG strains were obtained, which secreted the 37,000 fusion protein in their culture supernatant, which was confirmed by Western blot with specific immune serum against Ag85B and ESAT6. CONCLUSIONS: Recombinant BCG strains expressing Ag85B and ESAT6 fusion proteins of Mycobacterium tuberculosis were constructed. They may serve as new vaccine candidates for preventing tuberculosis.

[Mycobacterium tuberculosis secreting protein Ag85B-ESAT6 fused expression and purification].

OBJECTIVE: To investigate the fused expression of secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis, and to provide a promising preventive subunit vaccine against tuberculosis. METHODS: The gene encoding Ag85B and ESAT6 protein was amplified by PCR from genome of Mycobacterium tuberculosis H(37)Rv strain, and inserted into cloning vector P(GEM)-T-easy. After sequence analysis, and digestion by restriction endonuclease, Ag85B-ESAT6 was cloned into corresponding sites of the expression vector P(PRO) EXHT, and the recombinant plasmid was transformed into expressive strain E. coli DH5 alpha, induced with IPTG and fusion protein was purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with two fusion protein was detected by ELISA. RESULTS: The sequences of Ag85B and ESAT6 by PCR amplification were identical to those reported by GenBank. The recombinant plasmid fused expression protein of Ag85B-ESAT6 with relative molecular mass (Mr) of 37,000, which was confirmed by Western-blot analysis with specific monoclonal antibody against 6 x HismAb. The fused expression protein was insoluble. It could be purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with fusion Ag85B-ESAT6 was 1:1000 and that of mouse immunized with fusion protein ESAT6-Ag85B was 1:5000. CONCLUSIONS: Secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis was successfully fused expressed in E. Coli DH5 alpha. It may become a new type of vaccine against tuberculosis.

[Effects of Th1 cytokine gene on anti-CFP10 antibody production in BALB/c mice induced by Mycobacterium tuberculosis DNA vaccine].

AIM: To investigate the effects of plasmid containing mouse IL-12 and human IL-18 genes on the humoral immune response of mice immunized by CFP10 gene of Mycobacterium tuberculosis (MTB) H(37)R(v) strain. METHODS: Human IL-18 cDNA was amplified from RNA of PBMCs by RT-PCR and cloned into the pGEM-Teasy vector. After sequencing it was subcloned into the the sites of BamH I and EcoR I digestion of pcDNA3.1. BALB/c mice were injected intramuscularly by eukaryotic expression plasmid pcmIL12 and pcIL18, together with MTB CFP10 DNA vaccine, respectively. The same immunization repeated three times at intervals of two weeks. Mouse sera were collected at two weeks after the each injection. The titer of anti-CFP10 antibody was detected by ELISA. RESULTS: IL-18 cDNA was amplified successfully from RNA of human PBMCs by RT-PCR and the result of sequencing was correct. The IL-18 gene was correctly inserted into the vector pcDNA3.1 by being confirmed with BamH I and EcoR I digestion analysis, positive plasmid was called pcIL18. After being immunized with pcCFP10 three times, the end point titer of anti-CFP10 was 1:4 000, while the titer obtained by being immunized with pcIL18 + pcCFP10 was 1:8 000, but yet, after being immunized with pcmIL12+pcCFP10, the end point titer of anti-CFP10 antibody was only 1:200. CONCLUSION: Combination of IL-18 gene with MTB CFP10 DNA vaccine can enhance the humoral immune responses to pcCFP10, whereas the immunization with IL-12 gene plus pcCFP10 made humoral immune response markedly descent. As for whether IL-18 gene plus MTB CFP10 DNA vaccine can induce markedly the cellular mediated immune response to CFP10 or not remains to be further investigated.