[Objective] This study aimed to construct mammary gland-specific expression vector of bovine tracheal antimicrobial peptide (TAP) gene. [Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA sequence (NM_174776) in GenBank,and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRⅠand KpnⅠ, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein (EGFP) ,and transfected into bovine mammary epithelial cells ( bMEC) ,COS-7 cells and lactating rabbit mammary gland tissue by lipofectin transfection. The expression of green fluorescent protein in transfected cells was detected under fluorescence microscopy,and the expression of TAP mRNA in rabbit mammary gland tissue was detected by semi-quantity RT-PCR. [Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition,semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfected with pBLG-EGFP-TAP. [Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.
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