| The food safety problem is concerned by people all the time. It not only relates to the people's health and life, but also conserns the develepment of economy, the stability of society and the image of government and nation. The food-poisoning is the most commom problem of the food safety. And the bacterial-poisoning is most common in the food-poisoning problem. Vibrio is one of the most important pathogen that causes the food-poisoning of marine products. According to the report, there are at least twelve species in vibrio which can cause human's illness, such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio mimicus. In addition, it can do the damage to the sea economic animals, such as fish, crustacea and shell. That results in great damage to the sea cultivation. Therefore, the establishment of a kind of broad-spectrum, rapid methods to detect various species of food-poisoning Vibrio simultaneously is very important.The objects of our study were three toxin gene fragments, which respectively encode the mature proteins of the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus, the cytotoxin-hemolysin (VVC) of vibrio vulnificus and heat-labile hemolysin (VMH) of Vibrio mimicus. The PCR primers were designed and synthesized according to the gene sequences which were published in Genbank. And the three gene fragments tdh, vvhA and vmhA were amplified by PCR. Then the three toxin gene fragments were respectively ligated to the pMD 18-T Simple Vector, so as to construct the recombinant plasmids, pMD-18T-tdh, pMD-18T-vvhA and pMD-18T-vmhA. After the enzyme digestion and PCR identification, the three recombinant plasmids were sequenced and analyzed. The concordance of the sequencing results and gene sequences in GenBank is quite high: tdh 100%, vvhA 99%, vmhA 99%.PCR primers were designed with the flexible linker (GGGGS), then, the three toxin gene fragments were ligated in series by overlap extension PCR. After three PCR amplifications, the three-recombinant toxin gene (named FT) was amplified. FT was ligated to the pMD 18-T Simple Vector to construct the recombinant plasmids, pMD-18T-FT. After the enzyme digestion identification, the recombinant plasmids were sequenced. Then the software DNAStar 5.01 was used to analyze the sequencing results. The analytical results show that the concordance of gene sequence and GenBank′s was 99.6%. The sequence encoding the mature protein of the toxin gene was 3225bp, encoding 1074 amino acids. The mature protein weighted 120352.40Da with prediction. Then FT was subcloned into expression vector pET-22b(+). Recombinant expression vector 22b-FT was constructed followed by transforming into E. coli BL21 (DE3) for expression. The SDS-PAGE electrophoresis result indicated the molecular weight of the fusion toxin protein was about 120.4kDa in concordance with our prediction. The conditions of expression were optimizd. After 1mmol/L IPTG induction at 37℃for 4 hours, the fusion toxin protein expressed effectivelly in BL21 with the highest expression amount of 11.49% through thin layer scanning analysis. Most parts of the fusion toxin protein were cytorrhyctes.The cytorrhyctes of the fusion toxin protein were purified by the electroeluting to immunize cavia cobaya four times. On the seventh day after the fourth immunity, the blood was collected from the heart and the anti-serum was produced. The anti-serum value was 1?16 measured by the agar diffusion test. We used the anti-serum to establish a kind of indirect ELISA method for detecting food-poisoning Vibrio. The optimum working coonditions were determined: the working concentration of antigen was 2μg/mL, antigen was coated at 4℃overnight; the conditions of blocking was 1% BSA blocking for 90 minites; antibody was 1?16000 diluted,and reacted with antigen at 37℃for 60 minites; the enzyme-labeled antibody was 1?4000 diluted,and reacted with antibody at 37℃for 60 minites; substrate reacted for 10 minites. The indirect ELISA detection method was sensitive and specific through the sensitivity test, the specificity test and sample simulation test. The result of ELISA sensitivity test demonstrated that the indirect ELISA detection method's sensitivity degree to the three toxin were respectively: Vp TDH 0.03125μg/mL, VVC 0.0625μg/mL, VMH 0.03125μg/mL. The result of ELISA specificity test indicated that there were obvious positive reactions, when the antibody reacted with the three species of aim vibrio and toxin. But there was not any cross reaction, when the antibody reacted with three other species of vibrio and other twenty categories of bacteria.The indirect ELISA method for detecting food-poisoning Vibrio is broad-spectrum, is rapid, sensitive and specific. And it offers a new kind of method that can detect various species of food-poisoning Vibrio in food simultaneously. Moreover, it lays foundations of prepareing a rapid detection kit.
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