大肠杆菌O157:H7菌毛分子伴侣基因的克隆与表达

Cloning and expression of Escherichia coli O157:H7 fimbriae molecular chaperones gene

根据Genebank中已登录的大肠杆菌O157：H7菌株EDL933基因序列中菌毛分子伴侣ycbR基因序列设计1对引物，并分别在其5′端分别加入NcoⅠ、XhoⅠ酶切位点，以大肠杆菌O157：H7国内分离株97094的DNA为模板，用聚合酶链反应（PCR）扩增出710bp的DNA片段。回收并纯化该DNA片段，用限制性核酸内切酶NCOⅠ、XhoⅠ同时消化DNA片段和双酶切载体质粒pET28a（＋）。将它们回收纯化并连接，然后转化到宿主菌大肠杆菌DH5α，从该菌中提取重组质粒，用PCR、限制性内切酶位点分析及核苷酸序列测定法对克隆的重组质粒进行鉴定，表明ycbR基因定向克隆到了载体质粒pET28a（＋）。再将重组质粒转化到表达宿主菌大肠杆菌BL21（DE3），在含Kan抗生素LB培养基中经IPTG诱导12～16h，做SDS-PAGE分析，表明ycbR基因在菌株中获得高效表达，表达蛋白相对分子质量大小约为30000，与预期结果一致。为研究大肠杆菌ycbR基因产物的功能和致病作用奠定了基础。

To study the action of fimbriae molecular chaperones coded by ycbR gene, 710 bp DNA fragment was amplified by polymerase chain reaction （PCR） using a pair of primers designed on the ycbR gene sequence of EHEC strain ECL933 in Genbank and ended with Nco Ⅰ and Xho Ⅰ restriction endonucleases sites at their 5′ends. The PCR products amplified from EHEC strain 94H and the vector pET28a（＋） were digested with restriction endocleases Nco Ⅰ and Xho Ⅰ,ligated with T4 DNA ligase and transferred into bacterial host DH5α The recombinant plamid was further transferred into host strain BL21. The expression protein was detected by SDS-PAGE after 12-16 h with IPTG,showing that expected expression protein was about 30 000.