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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
关键词
奶牛
Β-防御素
LAP基因
绿色荧光蛋白
真核表达
dairy cattle
β-defensins
LAP gene
green fluorescent protein
eukaryotic expression
