Studies On Knockout, Expression And Regulation Mechanism Of Tdh2 Gene In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a Gram negative halophilic bacterium that is often found in seafood, salted food, marine and coastal environments. It is an important foodborne pathogen. Eating raw or undercooked seafood can cause human acute gastroenteritis and sepsis. TDH is considered as one of the main pathogenic factors of Vibrio parahaemolyticus, mainly encoded by tdh2 gene. Food poisoning caused by pathogenic Vibrio parahaemolyticus has become a focus of global food safety.studiess on the virulence gene-tdh of Vibrio parahaemolyticus are particularly important.In this paper, a suicide vector p DS132 was used in knockout of tdh2 gene of Vibrio parahaemolyticus RIMD2210633 by homologous recombination, and a tdh2 compensation strain was also constructed. Meanwhile, we compared and analyzed the biological differences between tdh2 mutant and the wild type strain. Based on the biological characteristics, the regulatory mechanisms of the virulence gene tdh2 of Vibrio parahaemolyticus were investigated. The research results will lay the foundation for the pathogenic mechanism of Vibrio parahaemolyticus and regulation mechanism of virulence genes.The main research contents are as following:1. Construction of tdh2 mutant and tdh2 compensation strain of Vibrio parahaemolyticusThe upstream and downstream flanking DNA fragments of tdh2 gene were fused by over-lap PCR, and then cloned into the suicide vector p DS132. The recombinant plasmid was introduced into Ecoli.S17-λpir and then transferred into Vibrio parahaemolyticus by conjugation. After two rounds of screening recombinant strains,tdh2 mutants were obtained. In order to verify whether tdh2 mutant had polarity mutation, we compensated tdh2 gene into tdh2 mutants. DNA fragments of tdh2 gene were obtained by PCR, and then cloned into the plasmid p ACYC184. The recombinant plasmid was introduced into tdh2 mutant by electrotransformation. After screening, tdh2 compensation strain was obtained.2. Biological characteristics of tdh2 mutantsMany reports indicated that the viabilities of pathogenic and non-pathogenic Vibrio parahaemolyticus in adverse environment were different. In order to investigate whether the tdh2 gene has impact on the viability of Vibrio parahaemolyticus in adverse environment, we analyzed the biological characteristics of the wild type strain and tdh2 mutant in growth, protein expression, antibiotics tolerance, resistance to acid and bile. The results showed that, no significant differences were found in the growth and antibiotics tolerance for the two type strains.However, the protein expression and resistance to acid and bile are significantly different. Proteins of the two type strains had obviously different expressions between29~44.3 KD, one of the different proteins was identified as bacteriocins C ABC transporter ATP-binding protein by LC-MS. Furthermore, WT was more resistant to acid and bile than tdh2 mutant.3. TDH expression and regulation mechanism of tdh2 gene in Vibrio parahaemolyticusTDH was expressed successfully through E. coli protein expression system.Furthermore, the real-time PCR was used to explore tdh2 gene regulatory mechanism preliminarily. The results showed that the m RNA expression levels of vp0301, vp1995,vp2553, vp1549, vpa1332, vpa1348, vpa1361, vp2890, vpa1312 and vpa313 gene in tdh2 mutant had a significant change compared to wild strain, which indicated tdh2 gene could regulate these genes.In this study, tdh2 gene was knocked out and the biological differences of tdh2 mutant and wild strain of Vibrio parahaemolyticus were compared. On this basis, tdh2 gene regulation mechanism was studied preliminarily by RT-PCR. The researchs will lay the foundation for the studies of pathogenesis and virulence gene regulation mechanism of Vibrio parahaemolyticus. Furthermore, the successful expression of TDH will be helpful for TDH research at the protein level and the development of engineering vaccine of Vibrio parahaemolyticus.