A Research For Risk Assessment Key Techniques And The Crystallization Condition Screening Of An Important Virulence Protein VopA Of Vibrio Parahaemolyticus

China is one of biggest countries in the production and consumption of aquatic products.According to research data,the seafood production and consumption of China have been increasing annually since 2001,and the production and consumption are ranked first in the world.Vibrio parahaemolyticus is the most common food-borne pathogen in aquatic products,which seriously influences the quality and safety of aquatic products,and limits the healthy and sustainable development of the aquatic industry.Microbial risk assessment is an important tool to control the harzard of V.parahaemolyticus in aquatic products.This research aimed at the deficiencies of hazard identification,exposure assessment and hazard characterization in the risk assessment of V.parahaemolyticus,and developed a new hazard identification technique for detection of V.parahaemolyticus based on viable bacteria DNA,established the stochastic models to describing the growth rate and bacterial concentration of V.parahaemolyticus based on the probabilistic distribution,and preliminarily studied the virulence protein VopA crystallography,which contributes to hazard characterization of V.parahaemolyticus.The main research contents and results are as follows:1.Research on a novel hazard identification technique for detection of V.parahaemolyticus based on viable bacteria DNAThe traditional V.parahaemolyticus hazard identification techniques was difficult to distinguish the dead and viable bacteria in aquatic products.Both of the number of dead and viable bacteria was usually calculated into the risk assessment,which easily leads to the wrong estimation of the infection risk,resulting in the uncertainty in risk assessment of V.parahaemolyticus.In order to more accurately identify the actual hazard of V.parahaemolyticus,it’s very useful to develop a novel hazard identification technique for detection of V.parahaemolyticus based on viable bacteria DNA.Propidium monoazide（PMA）is a photosensitive nucleic acid dye,in combination with qPCR technology,can selectively identify viable V.parahaemolyticus in aquatic products.The pretreatment process of PMA-qPCR involves two key steps:dark processing and photo-crosslinking.The traditional PMA pre-treatment steps are very complicated.In order to overcome this drawback,this study constructed a novel viable bacteria DNA screening device for PMA-qPCR pretreatment,which combined the two key steps of PMA pretreatment process.The results showed that the device can be used to set various PMA pre-processing conditions according to the requirements,including the dark processing time of PMA,light processing time and mixing speed.Through the real-time temperature data collection,there is no significant change in the temperature within 6 hours of continuous use of the device,which indicated it will be not harmful for the viable bacteria DNA in the sample.The device is fully automated,which simplifies the pre-treatment process of PMA-qPCR at the source,improves the processing efficiency of PMA.This device can be used for efficient screening of viable bacterial DNA,and provides a powerful tool for the hazard identification technique of V.parahaemolyticus.Based on the viable bacteria DNA screening device,combined with the advantages of PMA and multiple qPCR,this study developed a PMA-qPCR technique for detecting two common pathogens V.parahaemolyticus and Vibrio cholerae in aquatic products and aquatic environment.The results showed that the amplification efficiency of PMA-qPCR technique in this study can be maintained between 90%and 110%in different samples,and has a low limit of detection（LOD）.The LOD of this technique is 10¹-10² CFU/mL in the water of shrimp pond,and 10²-10³ CFU/g in shrimp and oyster.The method has a good ability to distinguish between dead and viable bacteria,and can accurately quantify viable bacteria of V.parahaemolyticus and V.cholerae in aquatic products even in the presence of10⁶ CFU/mL（g）dead bacteria.The technique was further applied to the detection of Vibrio spp.in real aquatic samples,and the results showed that the detection results of the technique were consistent with the ISO method.In this part of the study,a viable bacteria DNA rapid screening device for PMA-qPCR pretreatment was developed,and based on this device,a quantitative detection technique for Vibrio spp.in aquatic products was constructed,which to provides a new detection and analysis tools hazard identification of V.parahaemolyticus.2.Research on stochastic models of growth rate and concentration of V.parahaemolyticus based on probability distributionApplication of mathematical models to describe the bacterial growth rate and concentration were the two core components of exposure assessment in risk assessment.However,most models describing the growth rate and concentration of V.parahaemolyticus are deterministic models,and there is a lack of stochastic model studies for V.parahaemolyticus.Applying a probability distribution in exposure assessment to develop a new stochastic model will improve the accuracy of risk assessment of V.parahaemolyticus.This part of the study first collected the most comprehensive growth data of V.parahaemolyticus,and established a database for V.parahaemolyticus growth characteristics information.Based on the database,the data of the traditional model and molecular model of the growth of V.parahaemolyticus in shrimps were integrated to build a stochastic model.And the optimum probability distribution for describing the parameters of the square root model b and T₀ was investigated in this study.The results show that the probability distribution which is most suitable for describing the parameter b is Normal distribution,and its distribution fitting formula is Normal（0.0342,0.0068）.The optimum probability distribution for describing the parameter T₀ is the Uniform distribution,and its distribution fitting formula is Uniform（-9.7625,3.6925）.Based on this,the traditional square root model was modified to construct a novel stochastic model for describing the growth of V.parahaemolyticus in shrimps.Furthermore,a total of 992 seafood samples from major hypermarkets in Shanghai were monitored for prevalence and burden of V.parahaemolyticus from January 2011 to December 2012.In the meanwhile,appropriate probability distribution for describing V.parahaemolyticus concentrations was investigated based on these surveillance data.The results showed that the detection rate of V.parahaemolyticus（1.71%）and the geometric mean concentration of aquatic products in Shanghai large supermarkets were relatively low（0.1581 MPN/g）.There is no significant difference in the results of using lognormal distribution and integrated probability distribution to describe V.parahaemolyticus concentrations.The simple and effective lognormal distribution was recommended as a better choice for describing V.parahaemolyticus contamination in future exposure assessment.This part of the study developed a stochastic model for describing the growth rate of V.parahaemolyticus,and discussed the optimal probability distribution to describe V.parahaemolyticus concentrations,which provided useful tool for exposure assessment model of V.parahaemolyticus,which is beneficial for improving the accuracy of the risk assessment of V.parahaemolyticus.3.The expression,purification and crystallization conditions screening of the important virulence protein VopA for better understanding the hazard characterization of V.parahaemolyticusQualitative or quantitative analysis of harzard of pathogens is the key of hazard characterization in microbial risk assessment.The pathogenesis studies of V.parahaemolyticus will contribute to better understanding on the hazard characterization of this pathogen.VopA is an important virulence effector protein of V.parahaemolyticus type III secretion system,belonging to the YopJ family.The structure of VopA protein is still unknown,which greatly restricts the understanding of its pathogenesis.In this part of the study,the bioinformatics method was used to predict and analyze the physicochemical properties,transmembrane structure,signal peptide,secondary structure and tertiary structure of the key virulence protein VopA.The results showed that the VopA protein consisted of 289 amino acids with a molecular structure of C₁₄₁₀H₂₃₁₁N₄₀₁O₄₃₉S₂₀,a relative molecular mass of 32.55 KDa,a total atomic number of 4581.Th VopA is a hydrophilic protein with the average hydrophilicity of-0.404,and the absorption coefficient of VopA is 0.286 m²/mol.This protein has no significant signal peptide cleavage site and transmembrane region.The secondary structure is mainly composed ofα-helix and an extended chain.According to the tertiary structure analysis of VopA protein,the protein and the inositol hexaphosphate（IP6）may form a relatively stable crystal structure.Based on the bioinformatics analysis of VopA protein,the recombinant expression vectors 6His-MBP-TEV-VopA and 6His-TEV-VopA of VopA protein were constructed,which were introduced into E.coli BL21（DE3）respectively.And then the protein expression was induced by IPTG.Ultrasonic technology was empolyed to break up the cells,and extract the target protein in the supernatant.The VopA protein was extracted and purified by gel filtration.And then the crystallization conditions of the VopA protein were screened by using a protein crystallization screening robot.And then the protein crystallization conditions of VopA-IP6 complex were obtained in this study.The crystallization conditions of VopA protein were screened by using protein crystallization conditions screening robot combined crystallization kits.The protein crystallization conditions of the VopA-IP6 complex were obtained in the C3 well of the JCSG-plus^TM kit,which is 0.2 M ammonium nitrate,20%（w/v）PEG 3350.The further studies can be optimized to obtain higher quality protein crystals based on this crystallization conditions.This part of the study provides important data for revealing the structure and function of the effector protein VopA of V.parahaemolyticus type III secretion system II,which lays a foundation for the hazard characterization and pathogenesis of V.parahaemolyticus.Conclusion:This study aimed at the three aspects of hazard identification,exposure assessment and hazard characterization in microbial risk assessment,systematically developed a hazard identification technique for pathogenic Vibrio in aquatic products,and explored a stochastic model which can be used to improving the accuracy of exposure assessment,which provided the key technical support for the risk assessment of V.parahaemolyticus.And then the expression,purification and crystallization conditions screening of an important virulence effector protein VopA of V.parahaemolyticus were investigated in this study,which contributed to better understanding the hazard characterization and pathogenesis of V.parahaemolyticus.The conclusions of this study can provide technical and theoretical support for more effective assessment and control of the infection risk of V.parahaemolyticus,which will be beneficial for ensuring the healthy development of aquatic industry,improving the quality of aquatic products,and protecting the public health.