Isolation,ERIC-PCR Typing And Expressing Of Tdh Gene Of Vibrio Parahaemolyticus In Seafoods Of Shanghai

Vibrio parahaemolyticus, a halophilic gram-negative bacterium, is widely distributed in seawater, fish, shellfish and other seafood products. Pathogenic and non-pathogenic strains have been found. Pathogenic strains can cause acute gastroenteritis, such as diarrhea, vomiting, abdominal cramps and even death. Molecular distribution characteristics of Vibrio parahaemolyticus and rapid detection of this organism are of great importance.To investigate the prevalence of Vibrio parahaemolyticus,183samples were collected from freshwater fish farms in Shanghai, and195Vibrio strains were identified during2011-2013.195bacterial strains were identified to species, based on the sequencing of housekeeping gene gyrB, including137strains of V. parahaemolyticus(70.3%),24strains of V alginolyticus(12.4%),13strains of V. vulnificus (6.7%) and3strains of V. navarrensis (1.6%). PCR method was used to detect4virulence factors of V. Parahaemolyticus, including the thermostable direct hemolysin gene (tdh), the TDH-related hemolysin gene (trh), type secretion system structural protein gene (vcrDl and vcrD2). The result showed, vcrDl detection rate was100%, while the other3genes were tdh (2/137;1.5%), trh (0/137;0%) and vcrD2(1/137;0.74%) respectively. For strains from shrimps, crabs and shellfish, there was not significant difference in the distribution of4virulence factors.137strains of V. parahaemolyticus were typed using ERIC-PCR method. The electrophoresis results were digitalized by means of Image Lab and clusted by SPSS version21.0. All137strains showed good polymorphic fingerprinting map by ERIC-PCR method. One to fourteen bands could be amplified between100bp to4000bp by ERIC-PCR, typing137strains into6groups and16classes, while strains with same date and geographic location were mostly inclined to the same class. The ERIC-PCR classification method can be used as reference in the laboratory. It is simple and fast, and requires no template sequence compared to other molecular classification method, which is suitable for fast typing of a large number of strains. It is the suitable reference of the prevention of V. Parahaemolyticus and the epidemiological investigation. The theromostable direct hemolysin gene (tdh) was expressed in E. coli and the immunological activity of the recombinant protein TDH was analyzed. The result showed that it was successfully expressed as inclusion bodies with molecular weight of27kDa in E. coli. Western blotting analysis showed that the expressed protein maintain the same immunoactivity of natural protein; the rabbit polyclonal antibody to TDH was titrated by ELISA method and the titer was above1:256000. The expressed TDH can be used for the further research on immunological detection and rapid detection methods of V. parahaemolyticus.