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[目的]构建4种食源性致病菌融合毒素基因及重组表达载体,制备六联融合毒素的血清抗体。[方法]采用柔性Linker序列(G-G-G-G-S)对目的基因进行串联(HblA-VT1B-SEA-VT2B-BoNTaHc-SEB),构建重组表达质粒pET-22b(+)-F6并在E.coli BL21中进行表达,将表达蛋白纯化后免疫豚鼠制备血清抗体,利用ELISA和琼脂扩散试验验证抗体的特异性与敏感性。[结果]成功构建了重组表达质粒pET-22b(+)-F6并在E.coli BL21中成功表达,37℃表达蛋白主要以包涵体形式存在(表达量10.2%),基因序列全长3384bp,编码1127个氨基酸,蛋白分子量为127205,测序结果与设计序列一致性为100%。ELISA和琼脂扩散试验表明,融合毒素F6与4种食源性致病菌有良好的反应原性,与多种非目标菌不反应。[结论]成功构建了多联融合毒素基因的表达质粒及制备了抗血清,为利用融合毒素的方法检测食源性致病菌,进而建立食源性致病菌广谱、快速的检测方法奠定基础。
[Objective] The research aimed to construct four kinds of food-borne pathogenic bacteria fusion toxin gene and recombinant expression vector and prepare the serum of six-fusion toxin. [Method] HblA-VT1B-SEA-VT2B-BoNTaHc-SEB was constructed by using flexible Linker sequence (GGGGS) to construct recombinant expression plasmid pET-22b (+) - F6 and expressed in E. coli BL21 The expressed protein was purified and immunized guinea pigs to prepare serum antibodies. The specificity and sensitivity of the antibodies were tested by ELISA and agar diffusion test. [Result] The recombinant plasmid pET-22b (+) - F6 was successfully constructed and successfully expressed in E. coli BL21. The recombinant protein was expressed mainly in inclusion bodies at 37 ℃ (expression amount was 10.2%), and the full length of the gene was 3384bp. Encoding 1127 amino acids, the molecular weight of the protein is 127205, the sequencing result is 100% identical to the designed sequence. ELISA and agar diffusion tests showed that the fusion toxin F6 and 4 kinds of food-borne pathogens have good reactivity, and a variety of non-target bacteria do not respond. [Conclusion] The expression plasmid of polyclonal fusion toxin gene was successfully constructed and the antiserum was prepared. In order to detect food-borne pathogenic bacteria by using fusion toxin, a broad-spectrum and rapid detection method for food-borne pathogens was established basis.