绵羊透明质酸酶-2的真核表达及鉴定

Eukaryotic Expression and Identification of Hyaluronidase 2 in Sheep

为了构建绵羊透明质酸酶-2(hyaluronidase 2,Hyal-2)的真核表达载体并实现其在牛乳腺上皮细胞内瞬时表达,根据GenBank中绵羊Hyal-2基因序列(登录号:NM_001009754)设计2对引物,并分别在前段上游和后段下游引物5′端引入HindⅢ酶切位点和BamHⅠ酶切位点,同时在上游引物酶切位点后加入KOZAK序列,进行分段PCR扩增。以扩增的两段产物为共同模板运用重叠(overlapping)PCR技术扩增Hyal-2基因全长。通过连接反应将双酶切并纯化后的目的片段克隆于真核表达载体pEGFP-C1上,并通过脂质体转染法将构建好的真核表达载体转染牛乳腺上皮细胞,运用RT-PCR及West-ern blotting方法分别从核酸及蛋白质水平验证其表达。经测序分析,目的片段与模板核苷酸同源性为99%,氨基酸同源性为99%,且片段插入方向正确。RT-PCR及Western blotting方法检测均出现目的条带,证明成功构建了绵羊透明质酸酶-2的真核表达载体,并在牛乳腺上皮细胞中获得表达。

In order to construct of eukaryotic expression vector of hyaluronidase 2（Hyal-2） and its expression in bovine mammary epithelial cells,the full-length gene of Hyal-2 was amplified by overlapping PCR with two pairs of primers.Bringing HindⅢ restriction enzyme sites in upstream primer and BamHⅠ restriction enzyme sites in downstream primer.The expression recombiant plasmid was constructed by inserting the amplified gene into pEGFP-C1.The purpose gene was connected into eukaryotic expression vector pEGFP-C1 by connecting reaction.The vector was tranfected into bovine mammary gland epithelium cells by Lipofectamine 2000.The expression of Hyal-2 was verified by RT-PCR and Western blotting methods respectively.Sequencing analysis results showed that the nucleic acid homology was 99%,the amino acid homology was 99%.It was identified that the eukaryotic expression vector of Hyal-2 was successfully constructed and expressed in bovine mammary epithelial cells successfully.