The Study Of The Role And Mechanism Of Ipr1 Gene In The Macrophage Against Mycobacterium Tuberculosis

An estimated eight million people are infected each year with the pathogen Mycobacterium tuberculosis, and more than two million die annually. Yet only 10% of the infected people develop clinically manifest tuberculosis. Recently, 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, but no expressers die by necrosis, a messy cellular meltdown. Apoptosis is thought to help contain the disease by forming solid clumps called granuloma, while necrosis and associated inflammation help the bacteria multiply and spread between hosts. Ipr1 gene also improved resistance to another pathogen, Listeria monocytogenes, suggesting that it plays a general role in innate immunity. However, detailed mechanisms of how Ipr1 gene enhances the resistance of Mycobacterium tuberculosis in macrophages remain poorly understood.In this study, the Ipr1 full lengh coding sequence was obtained by RT-PCR from C57BL/6J mice thymus total RNA, a eukaryotic expression vector pEGFP-Ipr1 was constructed, and pEGFP-Ipr1 was transfected by Lipofectamine^TM 2000 into the murine macrophage cell line RAW264.7.The Ipr1 gene expression product on transcriptional level and translation level was detected, and the location of the expression product within the cells was identified. The influence of Ipr1 gene expression on macrophage growth and on macrophages anti-mycobacterial activity for H37Ra infection were observed. The gene chip technology was used to study the relationship between Ipr1 gene expression in macrophage and the relevant factors against intracellular pathogen infection.PartⅠConstruction and expression of the prokaryotic expression plasmid of Ipr1 geneObjective: To obtain full length coding sequence of Ipr1 gene and construct the prokaryotic expression plasmid carrying Ipr1 gene, express it in E.coli, detect the expression product.Methods: The coding sequence of the Ipr1 gene was amplified from the total RNA of C57BL/6J mouse thymus by RT-PCR , in which BamHⅠ,PstⅠrestrict endonuclease site in up and down primer was added respectively. The gene was cloned into pMD19-T simple-Ipr1, and the recombinant plasmid was identified by PCR, restrict endonuclease digestion and sequencing. A point mutation was repaired and then constructed a new plasmid include correct Ipr1 gene *pMD19-T simple-Ipr1. Insert the Ipr1 gene fragment into the prokaryotic expression plasmid pQE30 to obtain recombinant plasmid pQE30-Ipr1. Amplify the Ipr1 gene from *pMD19-T simple-Ipr1, add KpnⅠ,BamHⅠrestrict endonuclease site in up and down primer respectively, insert the Ipr1 gene fragment into another prokaryotic expression plasmid pET32a(+) to obtain recombinant plasmid pET32a(+)-Ipr1. The recombinant plasmid pQE30-Ipr1 and pET32a(+)-Ipr1 was transformed into E. coli, induced with IPTG, and analyzed by SDS-PAGE.Result: The whole coding sequence of Ipr1 gene was successfully amplified as expected. The accurate recombinant plasmid *pMD19-T simple-Ipr1 was identified by PCR, restrict endonuclease digestion and sequencing. Successfully constructed two prokaryotic expression plasmids pQE30-Ipr1 and pET32a(+)-Ipr1. After IPTG induction, we did not obtain the expression product of Ipr1 gene.Conclusion: Successfully obtain full length of Ipr1 gene from C57BL/6J mouse thymus. The prokaryotic expression plasmids pQE30-Ipr1 and pET32a(+)-Ipr1 were constructed successfully. But the expression of Ipr1 gene in E. coli has not been successful. This indicated that in E. coli the Ipr1 gene expression may be difficult.Part II The expression and location of Ipr1 gene in murine macrophage RAW264.7 cellsObjective: To construct a eukaryotic expression vector containing the fusion gene of mouse Ipr1 and EGFP and observe its expression in murine macrophage RAW264.7 cells.Methods: pET32a(+)-Ipr1 and pEGFP-C1 were digested by two restrict endonuclease respectively, then connected the Ipr1 gene fragment and pEGFP-C1 to construct pEGFP-Ipr1.The pEGFP-Ipr1 was identified by PCR, restrict endonuclease digestion. Then the pEGFP-Ipr1 was transiently transfected into RAW264.7 cells, and the expression of Ipr1 gene and fusion protein were detected by RT-PCR,Western blotting and laser scanning confocal microscopy.Results: The accurate eukaryotic recombinant plasmid pEGFP-Ipr1 was identified by PCR, restrict endonuclease. The the Ipr1gene expression product in the targeted cells was detected by RT-PCR,Western blotting. The fusion protein Ipr1-EGFP was localized in cell nucleusConclusion: The eukaryotic expression vector pEGFP-Ipr1 was successfully constructed. The fusion protein can be expressed in murine macrophage RAW264.7 and its localization was in cell nucleus.PartⅢThe influence of Ipr1 gene expression in murine macrophage RAW264.7 on cells'growth and in vitro anti-Mycobacterium capacityObjective:To detect whether Ipr1 gene expression influence the growth of the murine macrophage RAW264.7 and increase the anti-Mycobacterium activity of RAW264.7 cell.Methods:1.The pEGFP-Ipr1 was transiently transfected into RAW264.7 cells. We use MTT method to detect whether Ipr1 gene expression product inhibit the cell growth, and use TUNEL apoptosis method to observe whether the Ipr1 gene expression product can induce apoptosis by itself. 2. RAW264.7 cells were transfected with pEGFP-Ipr1 and the stable Ipr1 gene expressed RAW264.7 cells was selected by G418. 3. 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, then counted the CFU number of the cell lysatesResult: 1. Ipr1 gene expression product have no influence for cell growth, and even have no function to induce cell apoptosis. 2. The stable Ipr1 gene expressed cells was obtained after G418 selected. 3. In H37Ra infected macrophage, the CFU number in Ipr1 gene expression group is lower than that of control cells(p<0.05).Conclusion: Ipr1 gene expression product itself have no influence for cells growth and have no function to induce cells apoptosis. In vitro macrophage anti-Mtb test show that Ipr1 gene expression may increase the anti-Mtb activity. PartⅣThe impact of the immune response of the macrophages expressed Ipr1 gene against Mycobacterium tuberculosis infection by gene chip techniqueObjective: To study the effect and mechanisms of Ipr1 gene expressing in macrophages infected with Mycobacterium tuberculosis H37Ra by Oligo GEArray.Methods: The experimental group (Ipr1 +) RAW264.7 macrophages and the control group (Ipr1-) RAW264.7 macrophages were infected with Mycobacterium tuberculosis H37Ra. After 96h infection, the total RNA were isolated from the two groups. The cDNA and biotin-labeled linear cRNA were synthesized by RT-PCR, then purified the cRNA. After cRNA purification, the cRNA were hybridized to the Mouse Innate and Adaptive Immune Responses Microarray 113 genes. The chemiluminescence image were obtained by X-ray photo and scan. The differences in gene expression were analysised use software. Real-time quantitative PCR test was used to verify the reliability of the chip results.Result: Ipr1 gene expression up regulated 11 genes for macrophage anti-Mtb innate immunity involved TLRs signaling pathway: TLR2 and TLR4, Irak1 , Traf6 ; as well as interferon-related genes: Ifngr1 (IFN-γR1) and tumor necrosis factor-related genes: Tnfrsf1a (TNFR1),et al. And there was no significant difference about the molecular expression involved in the regulation of the adaptive immune response such as: IL-1, TNF-α, IL-12. The reduced expression of three genes Clecsf12, Il1rap, Ltf because of its low standard value (<0.05), its low accuracy may be of little value. The results of quantitative PCR reaction TLR2 and TLR4 and Ifngr1 indicated that the quantitative PCR results consistent with the trend of chip results.Conclusion: Based on the results of gene chip analysis, we initially speculated that the Ipr1 gene expression maybe enhance macrophage anti-Mtb infection by the mechanisms of increasing the innate immunity gene expression, in particular TLR2/TLR4 and its signal transduction molecules as well as the IFN -γR1, TNFR1 expression, and promote macrophage activation and intracellular anti-Mtb effect. This constitutes the basis for further study of the mechanisms of Ipr1 gene in host innate immunity against tuberculosis infection.