Expression And Preliminary Application Of The PPE68 Gene And Ipr1 Gene

In the study, we constructed a prokaryotic expression plasmid pET32a(+)-Ipr1, and we obtained the recombinant protein of Ipr1. Ipr1 gene and PPE68 gene of Mycobacterium tuberculosis were cloned into pBudCE4.1 to construct recombinant plasmid pbudce4.1-Ipr1-PPE68. The recombinant plasmid was transiently transfected into RAW264.7 cells. The expression of PPE68 gene and Ipr1 gene were detected by RT-PCR,Western blotting. Mycobacterium tuberculosis duplicon OriM gene was cloned into pbudce4.1-Ipr1-PPE68 to construct the shuttle-plasmid pBudCE4.1-PPE68-OriM-Ipr1. Electrotransformation the recombinant plasmid to BCG to construct PPE68 / Ipr1 recombinant BCG, and identified by colony PCR. And this provide an experimental basis for immunoprotection study of the PPE68 / Ipr1 recombinant BCG.Partâ… Construction and identification of the prokaryotic expression plasmid of Ipr1 geneObjective: To construct and identify of the prokaryotic expression plasmid of Ipr1 gene. Methods: The Ipr1 gene was amplified by PCR from pMD19-T simple-Ipr1, and cloned into pET32a(+) vector, then transformed into E. coli BL21. The recombinant proteins were expressed with IPTG induction. The rIpr1 protein was identified by SDS-PAGE and Western-blot.Result: Enzyme digestion and sequencing analysis confirmed that the recombinant plasmid pET32a(+)-Ipr1 was successfully constructed. The recombinant protein of Ipr1 gene was expressed in BL21.Conclusion: The recombinant plasmid pET32a(+)-Ipr1 was constructed successfully, The recombinant protein of Ipr1 gene in E.coli was also successful, which lays the foundation for prokaryotic expression of recombinant BCG of Ipr1.PART II Construction and identification of the eukaryotic coexpression plasmid containing PPE68 gene and Ipr1 geneObjective: To Construct a eukaryotic coexpression plasmid containing PPE68 gene of Mycobacterium tuberculosis and Ipr1 gene, and observe the co-expression of PPE68 gene and Ipr1 gene in murine macrophage RAW264.7.Methods: MTB PPE68 gene and Ipr1 gene were cloned into pBudCE4.1 which has multiple promoters to construct recombinant plasmid pBud68-Ipr1.The recombinant plasmid was transiently transfected into RAW264.7 cells, then we detected the expressions of PPE68 gene and Ipr1 gene by RT-PCR,Western blotting.Results: After by identifying restrict endonuclease ,the eukaryotic recombinant plasmid pBud68-Ipr1was condtructed successfully, and we detected the expressions of PPE68 gene and Ipr1 gene with RT-PCR,Western blotting.Conclusion: The recombinant plasmid pBud68-Ipr1 was constructed successfully. The protein can be expressed in murine macrophage RAW264.7, which lays the foundation for further development of PPE68/Ipr1 recombinant BCG vaccine against tuberculosis.Partâ…¢Construction and identification of PPE68 / Ipr1 BCGObjective: To construct and identify of PPE68/Ipr1 recombinant BCG.Methods: Mycobacterium tuberculosis duplicon OriM gene was cloned into pBudCE4.1-Ipr1-PPE68 to construct recombinant plasmid pBudCE4.1- Ipr1-PPE68-OriM and identified by PCR and enzyme digest. Electrotransformation the recombinant plasmid to BCG, and identified by coenobium PCR.Results: After identifying by PCR and restriction digestion, the shuttle-plasmid pBudCE4.1-Ipr1-PPE68-OriM was constructed successfully. The recombinant BCG was constructed successfully by coenobium PCR identification.Conclusion: The shuttle-plasmid pBudCE4.1-Ipr1-PPE68-OriM was constructed successfully. The PPE68/Ipr1 recombinant BCG was constructed successfully, which provides an experimental basis for immunoprotection study of the PPE68 / Ipr1 recombinant BCG.