大肠杆菌O157 Z4182基因的克隆与表达

Cloning and Expression of Escherichia coli O157 Z4182 Gene

根据大肠杆菌 O15 7Z4182基因设计 1对引物 ,并在其 5′端分别加入 Nco 、Xho 酶切位点 ,用聚合酶链反应(PCR)从本试验用的大肠杆菌 O15 7及 1株产志贺毒素大肠杆菌 (STEC) O8、1株肠道聚集粘附大肠杆菌 (EAgg EC)和 1株产肠毒素性大肠杆菌 (ETEC)菌株中也扩增出了 80 3bp的 DNA片段。以大肠杆菌 O15 7菌株 94H的 DNA为模板 ,用上述引物做 PCR,将其产物连接到载体质粒 p ET2 8a(+) ,然后转化到宿主菌大肠杆菌 L E392 ,从该菌中提取重组质粒 ,再将其转化到表达宿主大肠杆菌 BL2 1(DE3) ,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 Z4182基因定向克隆到了载体质粒 p ET2 8a(+)。将转化子培养并经 IPTG诱导后 ,做SDS- PAGE电泳 ,表明该菌株可以表达 Z4182基因。本试验为阐明大肠杆菌 O15 7Z4182基因的分布及探讨该基因产物的功能和致病作用奠定了基础。

Six strains of enterohemorrhagic Escherichia coli(EHEC) O157,seven strains of non-O157 Escherichia coli,two strains of Salmonella,two strains of Shigella,and two strains of Yersinia were detected by polymerase chain reaction(PCR) using a pair of primers designed on the Z4182 ge ne sequence of EHEC O157 and ended with NcoⅠ and XhoⅠ restriction endo nucleases sites at their 5′ends.The results indicated that all EHEC O157 strain s,one STEC strain,1 EAggEC strain and 1 ETEC strain were also positive for Z4182 gene.The PCR products amplified from EHEC strain 94H and the vector pET28a(+) w ere digested with restriction endonucleases NcoⅠ and XhoⅠ,ligated with T 4 DNA ligase and transferred into bacterial host LE392.The recombinant plasm id was further transferred into host strain BL21.Expression protein was detected b y SDS-PAGE.The induction tests with IPTG suggested that the 3 to 5 hours was th e most efficient for the expression of Z4182 gene.