Biological And Immunological Characteristics Research Of Recombinant Mycobacterium Smegmatis Expression Of Fusion Protein Rv2031c-Rv2626c

At present, third of the world’s population is infected with Mycobacteriumtuberculosis.China is a big burden country for tuberculosis (TB).A very importantreason of MTB become major of human pathogens is that MTB can cause latentinfection in the human body in a long time and the traditional anti-TB drugs onlyactivity when MTB growth. Bacillus calmette guerin (BCG) can’t protect TB inadult and can’t enhance the host immune response against latent infection.Therefore, it has important significance to research new TB vaccine to theprevention or treatment of latent infection of MTB for the control of TB.MTB could specific expresse dormant protein in the period of dormancy.These proteins are encoded by dormant regulator (DosR) genes, a total of48.MTB dormancy antigen with strong immunogenicity could interact with T cells from MTB persistent infection patients and promote T cell to release of IFN-γand IL-2. Two proteins of dormancy antigens, Rv2031c and Rv2626c play animportant role in the TB latent infection. They could abundantly express and aresign of latent infection. We know little about the function of them.Mycobacterium smegmatis (MS) is a kind of fast-growing non-toxic type ofmycobacteria, with good immunogenicity and genetic operability. It ishomologous with the MTB and could maintain a high degree of the spatialstructure and functional of antigen when expression of MTB antigens. So, weexpress Rv2031c and Rv2626c in MS as a TB vaccine could provide somereference of protein function and molecular mechanism of the latent infection.In this article we have constructed the recombinant Mycobacteriumsmegmatis expressing the fusion protein Rv2031c-Rv2626c and researched thebiological and immunological characteristics of recombinant Mycobacteriumsmegmatis (rMS).1. Construction and identification of recombinant Mycobacterium smegmatisexpressing the fusion protein of dormancy antigens Rv2031c and Rv2626c ofMycobacterium tuberculosis.To construct the recombinant rMS expressing the fusion protein of dormancyantigens Rv2031c and Rv2626c from MTB using an E.coli-Mycobacteriumshuttle expression vector. Rv2031c and Rv2626c genes was amplified from MTBH37Rv genome DNA by PCR and sequenced in the pMD18-T vector. The sizesof Rv2031c and Rv2626c gene fragments were435bp and480bp seperately, andthe sequences were identical to those of genes in the GenBank.The fusion gene of Rv2031c-Rv2626c was connected in the pMD18-T vectorusing enzyme restriction sites and then subcloned into the E.coli-mycobacteriumshuttle vector pDE22. The recombinant vector pDE22-Rv2031c-Rv2626c was transformed into MS by electroporation. The recombinant MS was analyzed thegene type by PCR, then induced at42°C, to analyze the expression of fusionprotein Rv2031c-Rv2626c by SDS-PAGE and Western-blot. The fusion genefragment of Rv2031c-Rv2626c could be amplified from the rMS by PCR. Thereslult of SDS-PAGE showed that the fusion protein Rv2031c-Rv2626c could bespecifically expressed in MS, with a relative molecular mass of35kDa. TheWestern-blot showed that the fusion protein Rv2031c-Rv2626c could be detectedby the monoclonly antibodies of Rv2031c and Rv2626c, and there was a specificbinding band appearred at the corresponding site.We used streaking inoculation method to research colony characteristics anddraw growth curves through of rMS measuring OD600.The results showed thatrMS colonies compared to MS were smaller and more rounded and the growthrate had no obvious changed.2. rMS interacte with murine macrophage.The rMS and MS infect murine macrophages RAW264.7with10:1MOI(bacteria: cell) in vitro.We use acid-fast staining, scanning electron microscopeand transmission electron microscope methods to study the infected macrophages,and use flow cytometry to measure macrophage apoptosis and necrosis. Infectedmacrophages were counted by cell counter and been lysed and counted by CFU.Culture supernatants were harvested at infection of macrophages with rMS andMS. The concentrations of cytokines in culture supernatants were determinedusing commercially available ELISA kits for IFN-γ、IL-6、TNF-α and NO. Theacid-fast staining results showed acid-fast stain is positive and the macrophagephagocytic mycobacteria.After infection of Macrophages for24h, count the number of livingcells.The results showed that the No. of intracellular bacteria of rMS group is higher than MS group，but the survival cell of rMS group is lower (P<0.05).Flow cytometry was used to measure apoptosis and necrosis ofmacrophage.The result of flow cytometry showed the apoptosis rate of rMSgroup was lower than MS group but the necrosis rate was higher (P<0.05).The concentrations of cytokines in were determined using commerciallyavailable ELISA kits for IFN-γ、IL-6、TNF-α and NO. The result of ELISAshowed rMS group induced the expression of IFN-γ, IL-6levels were higher thanMS group, but induce TNF-α and NO levels were lower (P<0.05).3. Immunological characteristics of rMS.MS, rMS and BCG were resuspended in phosphate buffered saline and weresubcutaneous inoculation by using1×107CFU per mouse.At4week afterimmunization, antibody content and subclass of antibody were measured byELISA.Lymphocyte proliferation index SI were detected by lymphocyteproliferation test and splenic lymphocyte CD4+/CD8+T cell ratio were detectedthrough flow cytometry.The concentrations of cytokines were determined usingcommercially available ELISA kits for IFN-γ, TNF-α, IL-2. The results ofantibody detection showed: anti Rv2031c and anti Rv2626c antibody from miceserum of rMS group was higher than MS, BCG and blank control group (P<0.05).Mouse serum antibody level of rMS group is higher than that of MS group andBCG group (P<0.05).Group of rMS induce the host to produce antibodysubclasses of IgG1was higher than that of IgG2a (P<0.05).Separate the splenic lymphocytes of components of immune mice,anddetecte splenic lymphocyte antigen specific proliferation index through WSTmethod.It showed that the proliferation of mouse splenic lymphocytes in rMSgroup (SI≥3) is higher than MS group and BCG group (P<0.05).Splenic lymphocyte CD4+/CD8+T cell ratio were detected through flow cytometry.The percentage of CD4+T cell for an average of25%,is higher than thepercentage of CD8+T cells for an average of14.5%(P<0.05).The concentrations of cytokines were determined using commerciallyavailable ELISA kits for IFN-γ, IL-2. The result showed that rMS group inducesthe expression of IFN-γ and IL-2was higher than group MS, group BCG andblank control group (P<0.05).In conclusion, the fusion protein Rv2031c-Rv2626c expression in MS,changed the biological and immunological trait of MS strains.The fusion proteincan promote RAW264.7cell expression of IFN-γ and IL-6, increased toxicity,promote necrosis, inhibition of apoptosis and the expression and secretion ofTNF-α and NO. In vivo immune experiment showed that, rMS could stimulatelymphocyte activation; stimulate host to produce specific antibodies; antibodysubclasses based on IgG1; stimulation of T lymphocytes to CD4+T celldifferentiation; promote more expression of IFN–γ and IL-2than MS.