奶牛乳腺炎金黄色葡萄球菌纤连蛋白结合蛋白配基结合区基因的克隆与原核表达

Cloning and prokaryotic expression of Fnbp D-domin gene from bovine mastitis Staphylococcus aureus

运用PCR方法扩增出奶牛乳腺炎分离株10A的Fnbp配基结合区基因,该片段大小为350bp左右,将其与PGEM-T载体连接,转化到受体菌DH5α中,用Amp/IPTG/X-gal琼脂平板筛选出含有重组子的菌株,鉴定成功后,测序结果表明:奶牛乳腺炎金黄色葡萄球菌10A的Fnbp配基结合区基因的序列与金黄色葡萄球菌Mu50、N315、MSSA476、MW2、RF122、COL以及NCTC8325的相应区域序列同源性分别为97%、97%、95%、95%、96%、94%和94%。将重组克隆载体质粒进行BamHⅠ和XhoⅠ双酶切回收后的DNA,定向插入到融合表达载体PET-32a中,构建了重组质粒PET-Fnbp,并转化到宿主菌BL21(DE3)株中,经IPTG4h诱导后,SDS-PAGE电泳表明,重组蛋白得到高效表达,重组蛋白主要以分泌形式存在,分子量35000左右,Ni柱纯化后的蛋白经Western-blotting检测,具有良好的抗原性和特异性。

Fnbp D-domin gene was amplified by PCR,whose templates came from bovine mastitis Staphylococcus aureus isolate 10A.The gene was about 350 bp,which was inserted into PGEM-T vector,then transformed to DH5α and screen with Amp/IPTG/X-gal agar plate.These recombinant plasmids were identified by PCR and restriction.The sequence of Fnbp D-domin indicated that:the homology rate was 97%、97%、95%、95%、96%、94% and 94% compared with Mu50、N315、MSSA476、MW2、RF122、COL and NCTC 8325 respectively.Fnbp D-domin gene of Staphylo...