中文摘要：

采用柔性Linker（GGGGS）和重叠延伸PCR方法将三个毒素基因,即副溶血性弧菌的耐热型直接溶血素基因tdh、创伤弧菌的溶细胞毒素基因vvhA和拟态弧菌的热不稳定型溶血素基因vmhA的成熟蛋白基因片段进行串联,得到三联融合毒素基因tdh-vvhA-vmhA（命名为FT）。一致性比对分析与预计融合的基因序列一致性为99.6%。FT的开放阅读框架全长3225bp,编码1074个氨基酸残基,预测分子量为120.4kDa。将FT亚克隆至表达载体pET-22b（＋）中,再转化至E.coliBL21（DE3）中进行表达,经SDS-PAEG分析,融合蛋白分子量与预测的相符。终浓度为1mmol/L的IPTG,37℃诱导4h,融合毒素蛋白表达量最高,经薄层扫描分析,此时融合毒素蛋白占全菌体蛋白的11.49%。融合毒素蛋白包涵体经纯化,免疫豚鼠制备血清抗体,确定间接ELISA的最佳工作条件,并进行敏感性、特异性、重复性及模拟样品检测试验,建立了食物中毒性弧菌广谱、快速、特异的同步免疫学检测方法。

英文摘要：

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 overla Pextension 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. Carla 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.