The Preliminary Study On The Apoptosis Of Mycobacterium Tuberculosis Secreted Protein Rv2031c And Rv2626c

Tuberculosis (TB) is a human beast of chronic infectious disease and a singlepathogen causing human death by Mycobacterium tuberculosis,the disease is seriousharm to human health.Various organs of the body can be invaded by Mycobacteriumtuberculosis,but mainly is TB.The main host of tuberculosis is human and animals,Itcan cause symptoms of cough,fever,coughing up blood after infection ofMycobacterium tuberculosis.The main reason of the major pathogen MTB can latentinfection in vivo persistence,Mycobacterium tuberculosis of Rv2031c and Rv2626cwere two key secreted proteins.The article from two main protein as start to begin,toconstruct eukaryotic expression vector and transfected cells, to explore the latency ofMycobacterium tuberculosis protein Rv2031c and Rv2626c impact on apoptosis, forfurther research on apoptosis of these two proteins impact and lay the foundationintracellular Mycobacterium tuberculosis survival mechanism.1.According to the nucleotide sequences of Mycobacterium tuberculosis H37Rvgenome published in GenBank,a pair of primers of Rv2031c、Rv2626c were designedand synthesized,Inactivated Mycobacterium tuberculosis H37Rv genome as atemplate,PCR amplification of full-length gene Rv2031c、Rv2626c to constructcloning vector pMD19-simple-T,The PCR product detected by PCR、double emzymeand company sequence.To construct recombinant plasmid named withpMD19-T-Rv2031c and pMD19-T-Rv2626c.respectively,pMD19-T-Rv2031c andpMD19-T-Rv2626c plasmid were digested by XhoI/PstI and XhoI/SalI, Rv2031cand Rv2626c gene fragments were recycled, respectively. respectively, and then theeukaryotic expression vector pEGFP-N1are connected by restriction analysis andDNA sequencing recombinant plasmid pEGFP-N1-Rv2031c, pEGFP-N1-Rv2626cplasmid correctness.The eukaryotic expression vector named pEGFP-N1-Rv2031cand pEGFP-N1-Rv2626c were constructed.2.pEGFP-N1,pEGFP-N1-Rv2031and pEGFP-N1-Rv2626c plasmid wereextracted by Endo-free Large-scale plasmid prep kit.The expression of the greenfluorescent protein was observed in the transfected293T cell24h byliposome-mediated transfection technique after transfection by fluorescencemicroscope.The three groups of cells total protein sample were analysised by westernblot.The western blot analysis of proteins Rv2031c-EGFP,Rv2626c-EGFP samemolecular weight, both the43KDa protein band.3.293T cells were transfected pEGFP-N1,pEGFP-N1-Rv2031and pEGFP-N1-Rv2626c three group plasmid,then detected flow cytometry of apoptosisrate at24h and48h.Meanwhile,total RNA was extracted and first-strand cDNAsynthesis,the apoptosis-related genes Bcl-2,Bax,Caspase-3and Caspase-8weredetected by real-time quantitative PCR methods at mRNA levels.Analysis of flowcytometry showed that the apoptosis rate of293T cells transfected withpEGFP-N1、pEGFP-N1-Rv2031c and pEGFP-N1-Rv2626c plasmid at24h and48h apoptosis rates were10%,21.8%,11.3%and13%,34.8%,15.8%.Analysis ofreal-time PCR indicated that the relative expression of Bcl-2gene graduallydecreased at24h and48h;But the relative expression levels of Bax,Caspase-3andCaspase-8increased significantly,a maximum at48h.Comprehensive analysis foundthat with pEGFP-N1-Rv2031c and pEGFP-N1-Rv2626c transfected into293T cell,itcould promote cell apoptosis,lay the foundation for further study of Mycobacteriumtuberculosis two proteins in intracellular survival mechanism.