The Development Of Nanobody And Construction Of Immunoassay Towards Vibrio Parahaemolyticus

Foodborne illnesses have serious impact on human health and are currently one of the public health problems that threaten human safety worldwide,with nearly one in ten people suffering from foodborne illnesses each year.Among foodborne pathogens,Vibrio parahaemolyticus（V.parahaemolyticus）,a Gram-negative rod-shaped bacterium widely present in coastal areas,can cause gastroenteritis in humans by consuming seafood contaminated with V.parahaemolyticus without proper treatment.Therefore,the rapid detection of V.parahaemolyticus plays an important role in preventing food poisoning and ensuring food safety.Colorimetric biosensors are widely used in environmental small molecule contaminant monitoring and food contaminant analysis because of their visualization,portability and low cost.Nanobodies are genetically engineered antibodies with small molecular weight,high specificity,high affinity,and easy access and expression,which are hot research topics in medical and food safety fields and have wide application potential as recognition elements in the construction of biosensors.In this study,a phage-displayed nanobody library for V.parahaemolyticus was constructed by immunization and genetic engineering,based on which three specific nanobodies were elicited and a sandwich ELISA was constructed for the detection of V.parahaemolyticus.Then,a colorimetric biosensor was developed using phage-Nbs to achieve the detection of V.parahaemolyticus with a single recognition element.The results of the study are as follows:1.A V.parahaemolyticus phage-displayed nanobody library was constructed,and V.parahaemolyticus nanobodies were obtained by panning.The V.parahaemolyticus antigen was prepared and used to immunize bactrian camel.Total blood RNA was extracted after immunization,and p Comb3X-VHH vector was constructed by genetic engineering and electrotransferred into receptor cells E.coli ER2738 to obtain a V.parahaemolyticus antibody library with a library capacity of 10⁷PFU,which was sequenced to show good library diversity.Three V.parahaemolyticus nanobodies were obtained by panning for target antigens Nb6,Nb20 and Nb28.2.A sandwich ELISA method was developed for the detection of V.parahaemolyticus in food.The above three antibodies were purified for expression.Nb20 and phage-Nb28were selected as pairwise antibodies to construct the sandwich ELISA.A checkerboard titration was applied to optimize the concentrations of the capture and detection antibodies,and the optimal concentrations of Nb20 and phage-Nb28 were obtained as 15μg/m L and5×10¹⁰PFU/m L,respectively.Ten common pathogenic bacteria were selected to study the specificity of the sandwich ELISA,and the results showed that the constructed method could only detect V.parahaemolyticus with good specificity.The detection limit of the method was 8.8×10⁵CFU/m L,which could be used for the detection of bacteria in shrimp samples.3.A colorimetric biosensor based on thiolated phage displaying nanobodies（phage-Nb-SH）was established to realize colorimetric and UV-Vis absorption spectroscopic determination.Phage-Nb20 was thiol modified and its binding ability was analyzed by laser scanning confocal microscopy（LSCM）and indirect ELISA.The results showed that phage-Nb20 remained its recognition activity after thiol modification.The reaction conditions of the colorimetric biosensor assay were optimized,and 3.8×10¹⁰PFU/m L was selected as the phage concentration and the reaction time was 100 min.The specificity of the colorimetric biosensor assay for ten common pathogenic bacteria was analyzed,and only V.parahaemolyticus could be detected.The detection limit of the method was 1×10³CFU/m L,and the recoveries were 97.9%～104.5%when applied to shrimp samples.In summary,this study established a phage-displayed nanobody library against V.parahaemolyticus,and three specific nanobodies were obtained after biopanning.A double-antibody sandwich ELISA and a single recognition element-based colorimetric biosensor was established,and successfully applied to the detection of shrimp samples.The colorimetric biosensor has a higher sensitivity than the sandwich ELISA analysis method,with a shorter detection period,which provided a possibility for the development of new technologies for the detection of foodborne pathogenic bacteria.