Study On The Apoptosis And Immune Efficacy Of Mycobacteirum Tuberculosis ESAT6/CFP10

Objective: Mycobacterium tuberculosis is a intracellular bacteria, the comparativegenomics study of BCG and H37Rv showed that the region of RD1was absent in allBCG strains and present in all Mycobacterium tuberculosis. It is hypothesized thatthe secretory proteins of ESAT6and CFP10in RD1region were related to cellapoptosis; BCG is the only vaccine of tuberculosis, but the protective effect was lowin adults. The eukaryotic expression plasmids of ESAT6and CFP10were used as aDNA vaccine and the mice were vaccinated with a combination strategy of BCG andDNA vaccine, then the antibody and the level of IFN-y were detected.Methods:(1) Primers of ESAT6and CFP10genes were designed，inactivatedmycobacterium tuberculosis H37Rv was used as templaet, full-length of ESAT6andCFP10genes were ampliifed by PCR to construct cloning vectors pGM18-T-ESAT6and pGM18-T-CFP10, respectively.(2) ESAT6and CFP10genes were subcloned topEGFP-Nl eukaryotic expression vector, named with pEGFP-N1-ESAT6andpEGFP-N1-CFP10, respectively;(3)293T cells was transfected with thepEGFP-N1-ESAT6or pEGFP-N1-CFP10, cell transfection results were observed bylfuorescence microscopy, and then the protein of cells were analysised by WesternBlot (4) The green fluorescent location of transfected293T and U937cells wereobserved by laser scanning confocal microscope.(5) Apoptosis rate of thetransfected cells were analysised by lfow cytometry, and then apoptotic genes relatedwith apoptosis were analysised with real-time PCR.(6) The mice wereco-immunized with pEGFP-N1-ESAT6(or pEGFP-N1-CFP10) and BCG. Their serawere collected for detecting the antibody and the level of IFN-y.Results:(1) The results of PCR, double enzyme digestion and sequencing showedthat pEGFP-N1-ESAT6and pEGFP-N1-CFP10were successfully constructed;(2)Cells transfected with pEGFP-N1-ESAT6(or pEGFP-N1-CFP10) plasmid expressedgreen fluorescent, analysis of Western Blot showed that ESAT6and CFP10proteinwere expressed successfully;(3) In293T cells transfected with pEGFP-N1-ESAT6(or pEGFP-N1-CFP10)，green lfuorescence was mainly distributed in cytoplasm; InU937cells transfected with pEGFP-N1-ESAT6, green lfuorescence distributed incytoplasm, however, the cells transfected with pEGFP-N1-CFP10， greenlfuorescence distributed in cytoplasm and partial region of the nucleus.(4) Analysisof lfow cytometry showed that the apoptosis rate of cells transfected withpEGFP-N1-ESAT6(or pEGFP-N1-CFP10) increased; Analysis of real-time PCRindicated that the relative expression of Caspase8gene was up-regulated in twogroup cells at12h,24h and48h; Caspase3gene up-regulated at48h only; Bcl-2gene down-regulated in two group cells at48h.(5) Antibodies of ESAT6(or CFP10)in the sera of mice co-immunized with pEGFP-N1-ESAT6(or pEGFP-N1-CFP10)and BCG can not be detected, but the level of IFN-y raised remarkably.Conclusions:(1) Recombinant eukaryotic expression vector pEGFP-N1-ESAT6(orpEGFP-N1-CFP10) of ESAT6(or CFP10) gene in Mycobacterium tuberculosis wereobtained.(2) The protein of ESAT6(or CFP10) could promote293T cells apoptosisafter transfection of pEGFP-N1-ESAT6(or pEGFP-N1-CFP10), and this may related to Caspase8，Bcl-2and Caspase3in the cells.(3)When pEGFP-N1-ESAT6(orpEGFP-N1-CFP10) was co-immunized with BCG，the levels of cellular immunityrised in mice.