Research Of Monoclonal Antibody To Thermostable Direct Hemolysin Of Vibrio Parahaemolyticus

This paper extracts and purifies thermostable direct hemolysin (hermostable direct hemolysin,TDH) of Vibrio parahaemolyticus,then studies on its relevant properties , and makes monoclonal antibodies of thermostable direct hemolysin (hermostable direct hemolysin,TDH) through hybridoma technology , and their properties are identified.Thermostable direct hemolysin (TDH) is secreted outside by Vibrio parahaemolyticus, using this characteristic this paper breeds pathogenic Vibrio parahaemolyticus in the special nutrient bacteria culture . Crude protein of TDH is got by salting-out,then after Several times chromatography in ion exchange chromatography and gel chromatography methods,eventually we purify the main pathogenic factor of Vibrio parahaemolyticus .Due to using TDH to immune animals below,and studying immunological methods to detect Vibrio parahaemolyticus, it is very important to research the properties of TDH.In this paper we study it from the denaturation temperatures, toxicity, heat resistance and several other physical-chemical correlation nature. Results show that it is typical of the protein. The toxin has strong toxic effects when injected into mice either intravenously or intraperitoneally in a small amount of TDH per mice. Meanwhile, this research also uses differential scanning calorimetry processing methods to determine the heat resistant of TDH , and proved the toxin has strong heat resistance. In this experiment, we get polycolonal antibodies of TDH through immuning mice. According to Western dot blotting principle,we establish an effective immune method for detection of pathogenic Vibrio parahaemolyticus. The working concentration of serum samples is drawed by chessboard assay. its specificity was judged through pathogenic Vibrio parahaemolyticusand no-pathogenic Vibrio parahaemolyticus.Results show that the samples with clear and obvious dots were judged as positive reaction Vibro parahaemolyticus was positive in pathogenic Vibrio parahaemolyticus,while no- pathogenic Vibrio parahaemolyticus was negative in the assay. The Western dot blotting was characterized in detecting pathogenic Vibrio parahaemolyticus by high sensitivity, simple operation and observable results.At present, monoclonal antibody(mAb) technology is widely used in bacteria testing field, and achieves good effect. We use TDH and joint adjuvants repeatedly to immune mices, according to hybrid tumor principle, which in turn by animal immune cell, fusion, monoclonal antibody(mAb) screening and clone, then study hybridoma cell types, the affinity, antibody specificity, stability properties, finally we succeed in making two mAbs of TDH, the experimental results show that the mAbs of TDH have strong affinity, high stability, high antibody specifility.In view of the close relationship between TDH and infection of pathogenic vibrio parahaemolyticus, we use the mAbs of TDH to do some preliminary explorations aboat applying in inspection prospects of Vibrio parahaemolyticus. The detection of Vibrio parahaemolyticus based on monoclonal antibody technology have great application prospect, and in the same time the mAbs of TDH lay a foundation for the development of colloidal gold technical inspection to detect pathogenic Vibrio parahaemolyticus.