奶牛LAP基因真核表达载体的构建及其在COS-7细胞中的瞬时表达  

Construction of eukaryotic expression vector of lingual antimicrobial peptide(LAP) gene from mammary gland tissue of cow and its transient expression in COS-7 cells

作　　者：曹随忠[1,2] 袁曦[1] 栾红雨[1] 余树民[1] 刘宗平[2] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国兽医学报》2011年第4期521-524,共4页Chinese Journal of Veterinary Science

基　　金：中国博士后科学基金资助项目(20090451250);扬州大学博士后基金资助项目(2008);四川省教育厅青年基金资助项目(09ZB054)

摘　　要：根据GenBank上发表的牛舌抗菌肽(Lingual antimicrobial peptide,LAP)基因cDNA序列设计1对特异引物,从患乳腺炎的奶牛乳腺组织中以RT-PCR方法扩增LAP基因片段,将其克隆到pMD19-T Simple载体中测序。重组质粒经EcoRⅠ+BamHⅠ双酶切回收目的基因片段,亚克隆入增强型绿色荧光蛋白表达载体pEGFP-C1中构建重组融合表达质粒pEGFP-LAP,将其脂质体转染COS-7细胞,经荧光显微镜观察到融合表达的绿色荧光蛋白,采用RT-PCR检测到LAP基因在COS-7细胞中转录。pEGFP-LAP重组表达质粒的成功构建为进一步研究奶牛LAP基因的表达特性及利用基因工程技术防治奶牛乳腺炎奠定了基础。The lingual antimicrobial peptide（LAP） gene of dairy cattle was amplified from the mammary gland tissue suffered mastitis by RT-PCR,using a pair of special primers according to the sequence published in GenBank,and then cloned into the pMD19-T simple vector.The LAP gene was extracted by digesting the recombinant plasmid,and subcloned into the pEGFP-C1 vector to construct eukaryotic expression vector named as pEGFP-LAP.Finally,the constructed plasmid pEGFP-LAP was transfected into COS-7 cells by lipofectin transfection,then the enhanced green fluorescent protein（EGFP） was observed under fluorescence microscopy,also the mRNA of LAP was detected by RT-PCR.The results indicated that the recombinant vector pEGFP-LAP was successfully constructed,providing an important subject for further investigation of expression feature of LAP and mastitis prevention by gene therapy.

关 键 词：奶牛 Β-防御素 LAP基因 绿色荧光蛋白 真核表达 

分 类 号：Q78[生物学—分子生物学]