金黄色葡萄球菌fnbB基因D2-D3编码区克隆及其植物表达载体构建

CLONING OF THE D2-D3 CODING SEQUENCE OF Staphylococcus Aureus fnbB GENE AND CONSTRUCTION OF ITS EXPRESSION VECTOR

金黄色葡萄球菌是引起奶牛乳腺炎的主要致病菌,其表面的纤连素结合蛋白(FnBP)在介导金黄色葡萄球菌侵入寄主细胞的过程中发挥着重要作用。因此,FnBP成为奶牛乳腺炎亚单位疫苗研究中的重要靶标。本研究根据Genbank中纤连素结合蛋白B基因(fnbB)D区编码序列设计引物,以金黄色葡萄球菌基因组DNA为模板,PCR扩增D2-D3区,将该片段回收后与pCAMBIA3301植物表达载体同时进行NcoⅠ和Eco 065Ⅰ(BstEⅡ)双酶切,酶切片段回收后进行连接,将连接产物转化大肠杆菌感受态细胞,通过菌落PCR、酶切鉴定等方法筛选阳性克隆,将阳性克隆送公司测序确定DNA序列。结果表明,阅读框架正确,氨基酸序列无突变,成功构建金黄色葡萄球菌fnbB基因D2-D3区植物表达载体。该研究为研制奶牛乳腺炎转基因植物口服疫苗奠定了基础。

Cow mastitis is mainly caused by Staphylococcus aureus. The fibronectin- binding protein （FnBP） of Staphylococcus au- reus plays an important role in the process mediating Staphylococcus aureus to invade host cells, and therefore, FnBP become one of the most important targets in the mastitis subunit vaccine research. In this study, firstly, the specific primers were designed according to the published sequence offnbB gene in Genbank and used to amplify the D2 - D3 coding region by PCR with genomic DNA of Staphylo-coccus aureus as template. The PCR products and the pCAMBIA3301 vector were digested by Nco I and Eco 065 I （ BstE II ） restriction enzymes and ligated by T4 DNA ligase. Then, the recombinant vector was transformed into E. coli DH5a and the recombinant colonies were identified by PCR and digestion. The results showed that the D2 - D3 coding sequence of Staphylococcus aureusfnbB gene was cloned and its expression vector p3301 D2 - D3 was constructed successfully. This study laid the foundation for the development of transgenic plant - based oral vaccine against cow mastitis.