Construction Of Eukaryotic Expression Vector Of Tuberculosis Resistant Gene Ipr1and Its Expression In Mouse Macrophages RAW264.7

In this study, the Ipr1full lengh coding sequence was obtained by RT-PCR fromC57BL/6J mice thymus total RNA, a eukaryotic expression of pEGFP-Ipr1was constructed,and pEGFP-Ipr1was transfected by LipofectamineTM2000into the murine macrophage cell.The Ipr1gene expression product on transcriptional level and translation level was detected,and the location of the expression product within the cells was identified.Part I: Construction and expression of the prokaryotic expression plasmid ofIpr1geneObjective: To obtain full length coding sequence of Ipr1gene and construct the prokaryoticexpression plasmid carrying Ipr1gene, express it in E.coli, detect the expression product.Methods: The coding sequence of the Ipr1gene was amplified from the total RNA ofC57BL/6J mouse thymus by RT-PCR, in which BamH I/EcoR I restrict endonuclease site inup and down primer was added respectively．The gene was cloned into pMD18-T-Ipr1, andthe recombinant plasmid was identified by PCR, restrict endonuclease digestion andsequencing. A point mutation was repaired and then constructed a new plasmid includecorrect Ipr1gene. pMDl8-T-Ipr1was perpared and digested, then ligated to plasmidpET32a(+) to obtain recombinant plasmid pET32a(+)-Ipr1. The recombinant plasmidpET32a(+)-Ipr1was transformed into E.coli, induced with IPTG and analyzed bySDS-PAGE.Result：The whole coding sequence of Ipr1gene was successfully amplified as expected．Theaccurate recombinant plasmid pMDl8-T-Ipr1was identified by PCR, cloned to pET32a(+),successfully constructed prokaryotic expression plasmids pET32a(+)-Ipr1. After IPTGinduction, we did not obtain of Ipr1gene expression product.Conclusion：Successfully obtain full length of Ipr1gene from C57BL/6J mouse thymus. Theprokaryotic expression plasmids pET32a(+)-Ipr1were constructed successfully. But theexpression of Ipr1gene in E.coli has not been successful. This indicated that the Ipr1geneexpression may be difficult in prokaryotic cell.Part II: The expression and location of Ipr1gene in murine macrophage cellsRAW264.7Objective：To construct an eukaryotic expression vector containing the fusion gene of mouseIpr1and EGFP and observe its expression in murine macrophage cells RAW264.7.Methods：pET32a(+)-Ipr1and pEGFP-C1were digested by two restrict endonucleaserespectively, then ligated the Ipr1gene fragment with pEGFP-C1to construct pEGFP-Ipr1.The pEGFP-Ipr1was identified by PCR, restrict endonuclease digestion. Then thepEGFP-Ipr1was transiently transfected into macrophage cells RAW264.7, and the expression of Ipr1gene and fusion protein were detected by RT-PCR, Western blotting and fluorescencemicroscope.Results：The accurate eukaryotic recombinant plasmid pEGFP-Ipr1was identified by PCR,restrict endonuclease. The Ipr1gene expression product in the targeted cells was detected byRT-PCR、Western blotting. The fusion protein Ipr1-EGFP was localized in cell nucleus.Conclusion：The eukaryotic expression vector pEGFP-Ipr1was successfully constructed. Thefusion protein can be expressed in murine macrophage and its localization was in cell nucleus.