Eukaryotic Expression and Identification of Hyaluronidase 2 in Sheep

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.