Preparation Of Specific Antibodies Against Vibrio Parahaemolyticus

Vibrio parahaemolyticus,a common food borne pathogens,is one of the most important micro-organisms in the recent years food poisoning incidents. It widely distributed in seawater, fish, shrimp, shellfish and other seafoods. Cardinal symptom of food poisoning caused by Vibrio parahaemolyticus are stomachache, diarrhea, vomiting, etc. Hemolytic toxin is the most important pathogenicity factor of Vibrio parahaemolyticus. TLH, TDH and TRH, which coded by tlh, tdh and trh genes respectively, are main hemolytic toxins of Vibrio parahaemolyticus.TLH is typical of Vibrio parahaemolyticus, while TDH and TRH are closed related with?pathogenicity of Vibrio parahaemolyticus. In order to supply materials for establishment of the immunological detection platform, in this study,recombinant hemolytic toxins of Vibrio parahaemolyticus and inactivated bacterial were used as immunogens to prepare the specific antibodies against Vibrio parahaemolyticus.1. Construction of the prokaryotic expression vectors of TLH, TDH and TRHThe prokaryotic expression vectors pET30a/tlh, pET30a/tdh, and pET30a/trh of hemolysin proteins TLH, TDH, and TRH were constructed by cloning and recombination, and transferred into E. coli BL21. His-TLH, His-TDH and His-TRH were expressed by IPTG induction successfully. 2. Preparation of monoclonal antibodies against Vibrio parahaemolyticusUsing the recombinant protein His-TDH as the sample of purification, the affinity chromatography method and gel extraction method were compared in purity, yield, and immunogenicity. The results show that gel extraction method is well in purity and has no effect with immunogenicity. Therefore, the gel extraction method was used to purify recombinant protein in this paper finally. After immunization, fusion, and screening, we obtained one specific monoclonal antibody against TLH and one specific monoclonal antibody against TDH. The two monoclonal antibodies are high specific against immunogens, but cannot be used to detect the culture of Vibrio parahaemolyticus.3. Preparation of polyclonal antibody against Vibrio parahaemolyticusThe bacterium, inactivated by ultraviolet lamp10min, was used as antigen. Polyclonal antibody was obtained after immunization, detection of titer, and purification. It was found that the polyclonal antibody can well identify Vibrio parahaemolyticus, while it has the weak response to Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa and an unknown strain of Vibrio by the ELISA detection.4. Preliminary discussion of application feasibilities of the antibodiesImmunomagnetic beads were used to analyze recombinant protein TLH and enrich Vibrio parahaemolyticus from sample. The results show that IMS-PCR method based on Vibrio parahaemolyticus polyclonal antibody obtained in this study, could achieve enrichment of Vibrio parahaemolyticus rapidly. Vibrio parahaemolyticus detection limit achieved approximately 10~3 CFU without condition of IMS-PCR optimized. And the immunochromatographic test strip base on TLH monoclonal antibody could achieve the quantitative detection of the recombinant protein TLH (detection limit is 0.01μg).