Construction and Identification of Mammary Gland-specific Expression Vector of Bovine Tracheal Antimicrobial Peptide (TAP)  

作　　者：Suizhong CAO Xueping YAO Yafei CUI Deying YANG Kang YONG Shumin YU Zongping LIU 

机构地区：[1]College of Veterinary Medicine,Sichuan Agricultural University [2]College of Veterinary Medicine,Yangzhou University

出　　处：《Agricultural Biotechnology》2013年第1期27-32,共6页农业生物技术（英文版）

基　　金：Supported by China Postdoctoral Science Foundation(20090451250);Youth Fund of Sichuan Provincial Department of Education(09zb054);Key Project of International Science and Technology Cooperation(2005DFA30720)

摘　　要：[ Objective] This study aimed to construct nmnmm_ry 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 se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and 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 mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue 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 transfeeted 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.[ Objective] This study aimed to construct nmnmm_ry 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 se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and 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 mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue 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 transfeeted 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.

关 键 词：Dairy cattle MASTITIS [3-defensin Trachea antimicrobial peptide （TAP） gene Manunary gland-specific expression vector 

分 类 号：S823[农业科学—畜牧学]