金黄色葡萄球菌聚集因子A(Clfa A)功能区基因的克隆及真核表达质粒构建  

Cloning and Construction of Eukaryotic Expression Plasmid of the Cluming Factor (clfa A) Functional Gene of Staphylococcus Aureus

作　　者：魏春梅[1,2] 杨宏军[1] 彭广能[2] 何洪斌[1] 王长法[1] 高运东[1] 仲跻峰[1] 

机构地区：[1]山东省农科院奶牛研究中心,山东济南250100 [2]四川农业大学动物医学院,四川雅安625014

出　　处：《家畜生态学报》2010年第4期69-72,共4页Journal of Domestic Animal Ecology

基　　金：山东省中青年科学家基金(BS2009NY002);山东省农业重大应用技术创新课题(2009);现代农业产业体系(nycytx-0107)

摘　　要：粘附素蛋白是金黄色葡萄球菌感染早期最重要的致病因子,研究根据金黄色葡萄球菌聚集因子A(clfa A)基因,设计合成特异性引物,以国内奶牛乳腺炎临床分离株金黄色葡萄球菌基因组为PCR模板,进行clfa A基因的克隆,并连接入pMD18-T载体进行测序和序列分析,然后经BamHⅠ和XbaⅠ双酶切后构建真核表达载体并进行鉴定。结果表明,以金黄色葡萄球菌标准株为对照,国内分离株多数在990 bp处扩增到特异性条带,经测序鉴定,与Genebank发表的金黄色葡萄球菌clfa A序列同源性为99%;构建的真核表达质粒通过PCR和双酶切鉴定证明构建成功,为研究核酸疫苗奠定基础。Adhesins are considered the most important virulence factors during early phases Staphylococcus aureus infection. To clone the gene of Cluming Factor （clfa A）, the staphylococcus aureus were isolated from the milk samples collected from dairy cows with peracute clinic mastitis. The DNA of the Staphylococcus aureus was extracted with classical technique to be template. The clfaA gene was amplified by PCR with the special primers designed according to the clfaA gene published in the GENEBANK. The product of the PCR was cloned into plasmid of pMD-18-T. The cloned gene was sequenced. A specific band was found in approximately 990bp, which accorded with the anticipative result. The amplified clfa A was 99% coincident with the gene of clfa A in the GENEBANK. After digestion by restriction enzyme BamH I and Xba I , the sequenced gene of clfaA was cloned into the pcDNA3.1 （＋）. The recombinant plasmid was transferred into competent E. coli and identified by restrition analysis. The result exhibited that the eukaryotic expression plasmid was constructed rightly which to the DNA vaccine against the infection of the Staphylococcus aureus.

关 键 词：乳腺炎 金黄色葡萄球菌 聚集因子A 

分 类 号：S811.5[农业科学—畜牧学]