牛源金黄色葡萄球菌的分离鉴定及ClfA基因的克隆和真核表达载体的构建

Construction of eukaryotic expression vector of ClfA gene from bovine Staphylococcus aureus

以研制安全有效的奶牛乳腺炎金黄色葡萄球菌基因工程疫苗为目的,采集380份隐性和临床型乳房炎奶样,经高盐肉汤、过氧化氢酶、甘露醇发酵、三糖铁发酵试验以及Baird-Parker平板对金黄色葡萄球菌进行分离和生化鉴定,PCR扩增分离株凝集因子基因(Clf A)并连接TA克隆载体。经测序确定无误后,连接pc DNA3.1His B以构建真核表达载体pc DNA 3.1His B-Clf A,转化Trans T1TM感受态细胞,HindⅢ和XhoⅠ双酶切分析,同时进行测序和序列分析。结果显示,通过高盐肉汤、过氧化氢酶、甘露醇、三糖铁试验和Baird-Parker平板从380份隐性及临床型奶牛乳房炎奶样中共分离到35株金黄色葡萄球菌,成功构建了真核表达载体(pc DNA3.1His B-Clf A),为研制金黄色葡萄球菌基因工程疫苗奠定基础。

The research developed a safe and effective genetic engineering vaccine of cow mastitis＇ Staphylococcus aureus. 380 milk simples from the cases of subclinic mastitis were isolated and characterized by salt meatbroth test, catalase test, mannitol fermentation test, triple sugar iron fermentation test and BP plat test,served as PCR templates for ClfA, then linked with the T vector for sequencing. Correctly cloned ClfA sequence were linked to the peDNA3. 1HisB, to construct eukaryotic expression vector pcDNA3. 1HisB-ClfA, i：ransformed TransT1^TM for sequencing and analysis,meanwhile HindⅢ and Xho Ⅰ double digestion to construct eukaryotic expression vector. The results showed 35 strains of Staphylococcus aureus were characterized from 380 milk sam- ples. The above test could be used for quick isolation of Staphylococcus aureus. The pcDNA3. 1HisB-ClfA was successfully constructed. This work would serve as the foundation for further researches of genetic engeering subunit vaccine for Staphylococcus aureus.