5种食源性致病微生物毒力基因重组质粒的构建、克隆表达与表达产物的研究

目的构建大肠杆菌O157∶H7、副溶血性弧菌、金黄色葡萄球菌、沙门氏菌和志贺氏菌5种主要食源性致病微生物毒力基因重组质粒载体并鉴定其表达产物,为探讨高通量磁性荧光纳米颗粒标记相应的抗体技术快速检测带毒力基因的致病微生物的可行性奠定基础。方法分别从5种食源性致病微生物毒力岛中选择特异性的毒力基因进行引物设计,分别提取5种目的细菌DNA片段,经PCR扩增、电泳回收目的基因片段,将目的基因片段与原核表达载体pET-23a、pET-28a、pET-32a构建各自的重组质粒,将重组质粒转化入大肠杆菌DH5α内并提取质粒载体,构建后的重组质粒进行酶切和测序鉴定,再转化入表达宿主E·coliBL21（DE3）。在0.1 mmol/L的IPTG诱导下,目的质粒载体在E·coliBL21（DE3）株中表达,SDS-PAGE电泳检测表达蛋白。结果实验成功构建大肠杆菌O157∶H7、副溶血性弧菌、金黄色葡萄球菌、沙门氏菌和志贺氏菌等细菌的毒力基因重组质粒载体pET-23a-eaeA、pET-28a-tdh、pET-28a-enterotoxin B、pET-32a-invA和pET-28a-ipaH,并克隆出969 bp的eaeA基因片段、567 bp的tdh基因片段、798 bp的enterotoxin B基因片段、1 041 bp的invA基因片段和771 bp的ipaH基因片段。重组质粒载体经酶切和测序与目标基因序列一致。在E·coliBL21（DE3）株中有质粒表达蛋白37.5 kDa（eaeA）、26 kDa（tdh）、34.5 kDa（enterotoxin B）、41 kDa（invA）、32 kDa（ipaH）。结论本研究成功构建了5种食源性致病微生物毒力基因重组质粒并在原核细胞中高效表达,为下一步获得相应的毒力基因抗体及快速诊断试剂的研制应用奠定了基础。

Construction,cloning expression and expression products of virulence gene recombinant plasmid in five food-borne pathogenic microorganisms

Objective To construct virulence gene recombinant plasmid vectors in five food-borne pathogenic microorganisms of Escherichia coli O157： H7, vibrio parahaemolyticus, staphylococcal aureus, salmonella, and shigella, identify their expression products, and explore the feasibility of directly detecting virulence genes of pathogenic microorganism by marking the corresponding antibody with high throughput magnetic fluorescent nanoparticles. Methods Specific virulence genes were selected for primer design from pathogenic islands in five food-borne pathogenic microorganisms, respectively. Firstly, DNA frag- ments were extracted from five target bacteria and gene fragments were obtained by using PCR and electro- phoresis. Then, respective recombinant plasmids, constructed by pET-23a, pET-28a and pET-32a with the gene fragments, were transformed into E · coli DH5α to extract plasmid vectors for enzyme and sequencing identification and transformed into E·coliBL21 （ DE3 ）. At last, the expression of target proteins were in- duced by 0. 1 mmol/L IPTG and analyzed by SDS-PAGE. Results Recombinant plasmid vectors pET-23a-eaeA, pET-28a-tdh, pET-28a-enterotoxin B, pET-32a-invA and pET-28a-ipaH of E ~ coli O157 ： H7, vibrio parahaemolyticus, staphylococcal, salmonella, and shigella were successfully constructed. The eaeA gene fragment of 969 bp, tdh gene fragment of 567 bp, enterotoxin B gene fragment of 798 bp, invA gene fragment of 1 041 bp, and ipaH gene fragment of 771 bp were cloned. The result showed that recombinant plasmid vectors were consistent with target gene sequence. Plasmid expressing proteins 37.5 kDa （eaeA）, 26 kDa（tdh） , 34. 5 kDa （enterotoxin B） , 41 kDa （invA） , and 32 kDa （ipaH） were detected in E ~ co- liBL21 （DE3） strain. Conclusion The virulence gene recombinant plasmids were successfully construc- ted in five food-borne pathogenic： microorganisms and efficiently expressed in prokaryotic cells, which laid foundation for obtaining corresponding virulence genes of antibod