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 BamH Ⅰ restriction enzyme sites in downstream primer. The expression recombiant plasmid was constructed by inserting the amplified gene into pEGFP-Cl. The purpose gene was connected into eukaryotic expression vector pEGFP-Cl by connecting reaction. The vector was tranfecled 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.%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BamH Ⅰ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.
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