Study On Haemolysin Activator Gene (eha) In Edwardsiella Tarda

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.