【作者】 成静；

【导师】 许化溪； 高大庆；

【作者基本信息】 江苏大学 ， 食品科学， 2007， 硕士

【摘要】 目的：迟缓爱德华菌(Edwardsiella trada，简称Et)是在水产养殖中重要的病原菌，能引起多种水产养殖动物，如鱼类、鳗鱼、龟鳖、牛蛙等的疾病。迟缓爱德华菌还是一种人畜共患病的病原菌，是爱德华菌属中唯一感染人的成员。预防和治疗水产养殖动物迟缓爱德华菌感染主要使用抗生素，这涉及到食品的安全问题。接种疫苗作为一种安全可靠的预防手段，已得到大家的公认，但目前尚无有效疫苗的应用。尽管已发现Et的一些重要的致病因子，如溶血素、载铁体、肠毒素等，但其致病机理仍不明确。因此，对迟缓爱德华菌的毒力因子及其调控的进一步研究，有助于发现新的致病机制和疫苗靶点。本实验旨在研究Et溶血素活化基因(Et haemolysin activator gene，eha)的生物学特性和调控作用，并构建其缺失株。方法：构建重组载体pET28a~+-eha，转化大肠埃希菌BL21，利用IPTG诱导大肠埃希菌表达EHA蛋白，菌体超声裂解物经Ni-NTA亲和层析柱纯化后，检测其纯化蛋白溶血活性及Vero细胞毒性。将eha重组质粒pED101和载体pACYC184分别转入大肠埃希菌DH5α中，利用RT-PCR及SDS-PAGE电泳，观察eha基因调控大肠埃希菌染色体上的细胞毒clyA基因的转录和表达。构建eha基因重组自杀质粒，利用同源重组原理，缺失Et的eha基因，并用PCR证实。结果：成功构建了pET28a~+-eha重组质粒，经IPTG诱导后，SDS-PAGE电泳显示表达出一条17 kDa左右的条带，纯化蛋白无溶血性及Vero细胞毒性。将eha重组质粒pED101转化入大肠埃希菌DH 5α中，其克隆子表现出溶血性，SDS-PAGE电泳显示克隆子有一条30 kDa左右的蛋白条带，与clyA蛋白分子量一致，RT-PCR显示其有约1000 bp的条带，也与clyA符合，而载体pACYC184转化入大肠埃希菌DH 5α中，其克隆子无溶血性，SDS-PAGE电泳和RT-PCR均未显示相应的条带。成功构建了eha基因重组自杀质粒，通过电转，将之转入Et中，得到了eha缺失株，用PCR证实Et eha缺失株中eha基因已部分缺失。结论：通过以上试验，初步证明了eha基因不是一个溶血素基因，而是一个调控基因，能够调控大肠埃希菌DH 5α中“沉默”的溶血素基因clyA的转录和表达。构建的Et菌的eha缺失株，为今后进一步探讨eha基因在Et菌中的致病机制的作用打下了基础。更多还原

【Abstract】 Objective: Edwardsiella tarda (Et) is an important causative agent in aquaculture. It has been responsible for diseases of all kinds of aquaculture animals such as fish,eel,turtle,bullfrog. Et is known to be a pathogenic bacteria for zoonosis and is only a pathogenic member in Edwardsiella for humans. Now, antibiotic is mainly used for preventing and healing Et infectious disease in aquatic products, but it is involved in a question about safe food and there is no effective vaccine for Edwardsiella. Pathogenesis of E. tarta infection is not well understood, although some substances such as hemolysin, siderophore, enterotoxsin ect have been reported as candidates for virulence factors in Et. The virulence factors of Et are further studied to find potential vaccine target and to understand Et pathogenic mechanism. In this experimental we want to study the biologic characteristic and regulating effect of eha and to construct its deleted strains(mutant strains).Methods: The recombination plasmid pET28a~+-eha was constructed andtransformed into E.coli BL21. Under the induction of IPTG, EHA protein was expressed in E.coli. The bacteria were broken by supersound and its product was purified through affinity column. The hemolysis effect of purified protein and its cytotoxicity to Vero cell was tested. The eha recombination plasmid pED101 and vector pACYC184 was respectively transformed into E.coli DH5α. With RT-PCR and SDS-PAGE electrophoresis, eha gene was tested to regulate the transcription and expression of cytotoxic clyA gene located on E.coli chromosome. The eha recombinated suicide plasmid was constructed and the eha gene was deleted from Et with homologous recombination. The deleted strains were proved with PCR.Result: The recombination plasmid pET28a~+-eha was successfully constructed.After IPTG induction, a band of approximate 17 kDa protein was found by SDS-PAGE electrophoresis. The purified protein was no hemolytic and no toxic to Vero cell. The eha recombination plasmid pED101 was transformed into E.coli DH5α. The clone was hemolytic and a band of above 30 kDa was shown with SDS-PAGE electrophoresis, A size of 1000 bp band was shown by RT-PCR. After the vector pACYC184 was transformed into E.coli DH5α, no hemolytic phenomenon was found and no propotional band was shown with SDS-PAGE electrophoresis and RT-PCR. The eha recombination suicide plasmid was successfully constructed. By electroporation transformation, it was transformed into Et .The deleted eha strains were got and were proved with PCR.From have been said above, this experiment proved initially that eha is not a hemolysin gene but a hemolysin- regulating gene. It can regulate the transcription and expression of silent hemolysin gene clyA in E.coli DH5α. The deleted eha Et strains had been constructed and were helpful to study the eha gene pathogenic mechanism in Et in future.更多还原