Construction of the Eukaryotic Expression Vector of Human Lysozyme and Expression in Bovine Mammary Gland Epithelial Cells

We constructed the eukaryotic expression vector pEGFP-C1-hLYZ and detected expression of human lysozyme in bovine mammary gland epithelial cells in vitro cultrue in order to do a research of transgenic donor cell for nuclear transfer in gene level.854bp human lysozyme gene(containing the open reading frame of 446 bp) was obtained by PCR amplification from human lysozyme plasmid.The PCR products were subcloned into pMD18-T vector.The fragments of recombinants were digested by Hind Ⅲ/BamHI and cloned into the eukaryotic expression vector pEGFP-C1,then transformed into Escherichia coli DH5α.A recombinant PEGFP-C1-hLYZ was constructed and confirmed by HindⅢ/BamHI digestion.Its expression was observed in 48 hours after it was transfected into bovine mammary gland epithelial cells.The eukaryotic expression vector pEGFP-hLYZ was successfully constructed and green fluorescence protein showing green fluorescence could be observed in mammary gland epithelial cells under fluorescent microscope.Human lysozyme gene was detected by PCR.