Abstract:To construct mouse Ipr1 gene (intracellular pathogen resistance 1)eukaryotic expression vector,the DNA fragment of Ipr1 gene was obtained through RT-PCR method from C57BL/6J mouse thymus.The gene fragment were cloned into simple vectors pMD19-T and transformed into E.Coli JM109.and the recombinant plasmid was identified by PCR,restrict endonuclease digestion and sequencing.Ipr1 gene was subcloned into pQE-Trisystem and the recombinant plasmid pQE-Trisystem-Ipr1 was comfirmed by restrict endonuclease digestion.Then the pQE-Trisystem-Ipr1 was transfected into RAW264.7,after transient transfection expression product of Ipr1 gene was detected by RT-PCR.Thus,the whole coding sequence of Ipr1 gene was successfully cloned as expected.A point mutation was found by sequencing.After the point mutation was repaired and the accurate recombinant pQE-Trisystem-Ipr1 was obtained,Ipr1 gene expressed in transfected RAW264.7 was detected by RT-PCR.Through these procedures,the coding sequence of Ipr1 gene was obtained from C57BL/6J mouse thymus and the eukaryotic expression vector for Ipr1 gene was successfully constructed.This laid the foundation for the further stndy on the funetions of Ipr1 gene.
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