中国生物工程杂志

To obtain the tervalent fusion toxin gene (named FT), three toxin gene fragments from three species of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio mimicus were connected with the flexible linker (GGGGS) using overlap extension PCR. The three toxin gene fragments respectively encode the mature proteins of the thermostable direct hemolysin (TDH) of V. parahaemolyticus, the cytotoxin (VVC) of V. vulnificus and the heat-labile hemolysin (VMH) of V. mimicus. The identity of FT nucleic acid sequence was 99.6% with the corresponding toxin gene fragments. The open reading frame of FT was 3225 bp, encoding 1074 amino acid residues with the predicted molecular weight (MW) of 120.4 kDa. Then, FT was subcloned into the expression vector pET-22b(+). The construction of recombinant expression vector pET-22b-FT was followed by transforming into E. coli BL21(DE3) for expression. The SDS-PAGE electrophoresis results indicated that the MW of the fusion toxin protein was matched to the predicted MW. After induction by 1 mmol/L IPTG at 37℃, the fusion toxin protein was effectively expressed in E. coli BL21(DE3) with the amount of 11.49% through thin layer chromatography scanning (TLCS) analysis. Cavia cobaya was immunized using the purified cytorrhyctes to produce the anti-serum. Through the determination of the optimum working conditions, the sensitivity test, the specificity test, repeatability test and sample simulation test, the indirect ELISA method was established, which is a broad-spectrum, rapid and specific to detect various of food-poisoning Vibrio simultaneously.