The Study Of Macrophage Ipr1 Function For The Mechanism Of Innate Immunity Against Tuberculosis

In 2005 researchers have found a gene Ipr1(intracellular pathogen resistance 1) in mice that plays a major role in containing Mycobacterium tuberculosis.The macrophages that express Ipr1 gene tend to die in a controlled manner called apoptosis which control Mtb multiply,while no expressers die by necrosis which help the bacteria multiply and spread between hosts.However,detailed mechanisms of how Ipr1 gene enhances the resistance of Mycobacterium tuberculosis in macrophages remain poorly understood.In this study,the Ipr1 coding sequence was obtained by RT-PCR from C57BL/6J mice thymus,a eukaryotic expression vector pEGFP-Ipr1 was constructed to observe the influence of Ipr1 gene expression on macrophages anti-mycobacterial activity for H37Ra infection.We construct the eukaryotic coexpression shuttle plasmid pBGOI,and transform it into BCG,Then the expression of the recombinant BCG which called BCGi was demonstrated in vitro and in vivo.The mice of C3HeB/FeJ were immunized with BCGi to observe the influence of BCGi against infected mice with Mtb and to analyse the mechanisms by the technology of Gene Chip,Protein Chip,ELISA,Real-time PCR.PartⅠThe influence of Ipr1 gene expression in murine macrophage RAW264.7 on anti-Mycobacterium capacity in vitroObjective:To obtain full length coding sequence of Ipr1 gene and construct a eukaryotic expression vector containing the fusion gene of mouse Ipr1 and EGFP and observe its expression and location in murine macrophage RAW264.7 cells and observe whether Ipr1 gene expression increase the anti-Mycobacterium activity of RAW264.7 cells.Methods:The Ipr1 gene was amplified from the total RNA of C57BL/6J mouse thymus by RT-PCR,The gene was cloned into pEGFP-C1 to construct pEGFP-Ipr1 identified by PCR,restrict endonuclease digestion.Then the pEGFP-Ipr1 was transiently transfected into RAW264.7 cells,and the expression of Ipr1 were detected by RT-PCR,Western blotting and laser scanning confocal microscopy.RAW264.7 cells were transfected with pEGFP-Ipr1 and the stable Ipr1 gene expressed RAW264.7 cells was selected by G418.Experimental group macrophage cells(Ipr1+) and control cells(Ipr1-) were infected by M.tuberculosis strain H37Ra respectively.After 24h and 96h infection we cultured the cell lysates on 7H10 medium,and counted the CFU number of the cell lysatesResult:The whole coding sequence of Ipr1 gene was successfully amplified as expected.The eukaryotic plasmid pEGFP-Ipr1 was constructed and the Ipr1 gene expression product in RAW264.7 cells was detected by RT-PCR,Western blotting.The fusion protein Ipr1-EGFP was localized in cell nucleus The stable Ipr1 gene expressed cells was obtained after G418 selected.In H37Ra infected macrophage,the CFU number in Ipr1 gene expression group is lower than that of control cells(p＜0.05).Conclusion:Successfully obtain Ipr1 gene from C57BL/6J mouse thymus.The pEGFP-Ipr1 was successfully constructed.The fusion protein can be expressed in murine macrophage RAW264.7 and its localization was in cell nucleus.In vitro macrophage anti-Mtb test show that Ipr1 gene expression may increase the anti-Mtb activity.PartⅡThe construction and Identification of recombinant BCG (BCGi) targetedly deliver pBGOI into macrophages.Objective:To construct the eukaryotic coexpression shuttle plasmid pBGOI and express it in A549 cells.To construct a Ipr1 recombinant BCG (BCGi) strain that can targetedly deliver the eukaryotic coexpression plasmid pBGOI encoding Ipr1 into macrophages.To observe the expression of BCGi in vitro and in vivo.Methods:Coding sequences of Ipr1,GFP,and OriM were simultaneously cloned into pBudCE4.1,which had multiple promoters to form the shuttle plasmid pBGOI.The A549 cells were transfected with the formed plasmid by Lipo-fectAMINE2000,and the expression of Ipr1 and EGFP was detected by RT-PCR,immunohistochemistry,Western Blotting, fluorescence microscope.The shuttle-plasmid pBGOI was transformed into BCG and identified by PCR.The Ipr1 and GFP expression of recombinant BCG which named BCGi were detected by RT-PCR, immunohistochemistry,Western Blotting in vitro and in vivo.Results:Restriction enzyme digestion and sequence analysis showed that pBGOI had been constructed successfully.Ipr1 protein was detected in the transfected A549 cells,meanwhile the expressed EGFP was observed under fluorescence microscope.The recombinant BCG was successfully constructed.The expression of GFP and Ipr1 was detected by RT-PCR and immunohistochemistry in vitro and in vivo infected with the recombinant BCGiConclusion:The eukaryotic coexpression shuttle vector named pBGOI was successfully constructed,and the recombinant BCGi was successfully constructed,it provides a good basis for further research on the function and mechanisms of Ipr1 against tuberculosis.PartⅢThe influence of Ipr1 gene expression in vivo and the probable mechanisms against tuberculosis.Objective:To observe the influence of BCGi which can targetedly deliver Ipr1 gene into the mice macrophages against infected mice with Mtb and to analyse the probable mechanisms.Methods:the C3HeB/FeJ mice infected with Mtb were immunized with BCGi.after 3 weeks,the mice were killed.Then the numbers of viable bacteria in the lung and spleen were counted.Lungs and spleens were observed for pathological analysis.The expression of Ipr1 in tissue was detected with immunohistochemistry.Gene chip and Real-time PCR of lung,protein chip of serum were detected.The levels of IL-10, TNF-α,IFN-γin serum were detected with ELISA.Result:a significant reduction in the number of Mtb organisms was achieved in BCGi group.The Pathological changes of the lungs in BCGi group were better than BCG group and PBS group.The expression of Ipr1 in lungs was found.BCGi group gene expression up regulated 13 genes: Igh-6,Lbp,Ltf,Ly96,Ncf4,Nfkb1,Nfkb2,Nfkbia,Nos2,Prg2,Sftpd,Stab1,Tlr4。Fas,Mcl1,Bcl2,Caspase3,iNOS,LRG47,NRAMP1 genes expression were up regulated in the results of quantitative PCR reaction。The reduced expression of antibody in serum were:Eotaxin,Eotaxin-2,IL-1β,IL-3,IL-6,IL-10,IL-17,I-TAC,Leptin,TNFα。The serum levels of IFN-γin BCGi group detected by ELISA was higher than that of BCG group。Conclusion:The recombinant BCGi had effects against tuberculosis infection.The Ipr1 gene maybe enhance macrophage anti-Mtb infection by the mechanisms of increasing the innate immunity gene expression,in particular TLR4 and its signal transduction molecules expression.