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.

[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.

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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.