Virulence Gene Detection Of Vibrio Parahaemolyticus From Litopenaeus Vannamei And Cloning, Expression Of Tlh Gene

Vibrio parahaemolyticus is one of the important pathogen in aquaculture of coastal areas of China, widely distributed in seawater and seafood products. Aquatic animal diseases caused by explosive proliferation of V. parahaemolyticus have brought huge economic losses every year. The microbial foodborne disease caused by contamination of V. parahaemolyticus tops the list in China. Guangxi is located in the south of China, where the seafood is consumed widely and people like eating raw or undercooked seafood. Therefore V. parahaemolyticus infection is very common in Guangxi. Thermostable direct hemolyticus (TDH), TDH-related hemolysin (TRH) and Themolabile hemolysion (TLH) are the most important virulence factors of V. parahaemolyticus, which can produce a variety of hemolysins. The three virulence factors are encoded by tdh, trh and tlh genes, respectively. In this study,160 samples of Litopenaeus vannamei were collected from Qinzhou, Beihai and Fangchenggang of coastal Guangxi firstly. Then strains of V. parahaemolyticus were isolated, identified and checked the antimicrobial susceptibility. Virulence genes of tdh, trh and tlh were detected by PCR method. Finally, the tlh gene was successfully expressed, which lays the foundation for the protein purification, antibody preparation and protein functions, and provides a platform to monitor and prevent the infection of V. parahaemolyticus. The results were summarized as follows:1. Sixty-seven strains of V. parahaemolyticus were isolated from 160 samples of L. vannamei, according to the conventional bacterial isolation and identification based on API analysis and 16S rRNA sequencing. The detection rate was 41.88%.16S rRNA gene sequences of the 67 strains were tested with BLAST analysis in GenBank. The results indicated that the tested sequences show high homologies (similarity rate around 98.8%～100%) with the standard strain of V. Parahaemolyticus viz. ATCC17802 (NRl 14632.1). Drug sensitivity tests showed that 67 strains are sensitive to 12 kinds of antibiotics viz., fortum, ceftriaxone, sulfamethoxazole trimethoprim, sulperazone, norfloxacin, enrofloxacin, ciprofloxacin, levofloxacin, enoxacin, ofloxacin, tazocin and florfenicol, and that the sensitive rates are all above 80%. Besides, the 67 strains were resistant to penicillin G, ampicillin, Sulphamethazine, sarafloxacin hydrochloride and sulfanilamide-6-methoxamine pyrimidine and the drug resistance were great.2. Virulence genes tdh, trh and tlh were detected by PCR method in 67 strains of Vibrio parahaemolyticus. Results showed that gene tlh was positive in all the V. parahaemolyticus strains while gene tdh and trh were all negative in all strains.3. Gene tlh was cloned from the Vibrio parahaemolyticus by PCR, and the cloned fragment was ligated into prokaryotic expression vector pET-28a of Escherichia coli. Then the recombinant plasmid pET-28a-tlh was transformed into E. coli BL21 and the recombinant proteins were expressed by induction of IPTG. The expression products were analyzed by SDS-PAGE and the results showed that the size of expression products corresponds to the estimated size of 61KDa. Western blotting results showed that recombinant protein TLH could reacted specifically with Anti-6×His tagged antibodies, which demonstrated the accurate expression of the recombinant protein TLH in E. coli prokaryotic expression system.