副溶血性弧菌融合鞭毛基因的表达与免疫学性质

Expression and immunological character of poly-recombinant flagellae gene for Vibrio parahaemolyticus

采用柔性Linker序列(Gly4Ser)对目的基因进行串联(FlaA-FlaF-FlaB),构建重组表达质粒pET-22b(+)-F3并在E.coliBL21中进行表达,将表达蛋白纯化后免疫獭兔制备血清抗体,利用ELISA和琼脂扩散试验验证抗体的特异性与敏感性。结果显示,成功构建了重组表达质粒pET-22b(+)-F3并在E.coliBL21中得到表达,表达蛋白主要以包涵体形式存在(表达量23.5%),基因序列全长3420bp,编码1140个氨基酸,蛋白相对分子质量为119000,测序结果与设计序列一致性为99%。ELISA和琼脂扩散试验表明,融合鞭毛F3与其他几种中毒性弧菌鞭毛具有一定的免疫交叉性,与多种非目标菌不发生反应。从而表明,多联融合鞭毛基因的表达质粒已构建成功并制备了抗血清,为利用融合鞭毛的方法检测食物中毒性弧菌,进而建立食物中毒性弧菌广谱、快速的检测方法奠定了基础。

Vibrio parahaemolyticus poly-recombinant flageUae gene and recombinant expression vector have been constructed, then the serum antibody of poly-recombinant flagellae prepared. Use the flexibility hnker（Gly4Ser） to connect the purpose gene （FlaA-FlaF-FlaB）,then construct recombinant plasmid pET-22b（ ＋ ）-F3 and carry out expression in E. coli BI21. The rabbits were immunized using the expression protein after purification to get serum antibody. The results of ELISA and agar diffusion reaction indicated the specificity and sensitivity of the antibody. The recombinant expressing plasmid pET-22b（ ＋ ）-F3 was constructed and expressed in E. coli BI21 successfully. The greater part of expression protein is inclusions with the expression amount of 23.5%, encoding 1 140 amino acids with the molecular weight of 119 000, and total long of 3 420 bp, and the concordance of gene sequence corresponding design＇s is 99%. The ELISA and agar diffusion reaction indicated the poly-recombinant flagellae could product different immune intersect reaction with other food-poisoning Vibrios but did not react with some no-objective bacterium. Recombinant expression plasmid of poly-recombinant flagellae gene was constructed and the serum antibody prepared successfully. It may be used to check food poisoning bacteria and establish the board-spectrum, quick, special detecting way.