人溶菌酶基因真核表达载体构建及其在牛乳腺上皮细胞中的表达

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

构建真核表达载体pEGFP-C1-hLYZ,并从基因水平研究人溶菌酶基因在体外培养的牛乳腺上皮细胞中的表达,为核移植提供转基因供体细胞。采用PCR的方法从人溶菌酶质粒中扩增854 bp人溶菌酶基因(包括完整的编码框446 bp)。并克隆到pMD18-T载体上,用限制性内切酶HindⅢ和BamHⅠ双酶切。将人溶菌酶基因的酶切片段(883 bp)定向克隆到pEGFP-C1的多克隆位点中,构建重组表达质粒pEGFP-hLYZ,并进行HindⅢ和BamHI双酶切鉴定。转染体外培养的牛乳腺上皮细胞,48 h后观察其表达情况。PCR检测溶菌酶基因的表达。成功构建了人溶菌酶真核表达载体,观察到表达绿色荧光蛋白的牛乳腺上皮细胞,并检测到溶菌酶基因。

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 Hind Ⅲ/ 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.