[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] To construct toxin including fusion toxin gene from food poisoning bacteria and its recombinant expression vector,and then prepare serum antibody of Six-valent fusion toxin.[Methods] Six gene(HblA—VT1B—SEA— VT2B—BoNTaHc—SEB)fragments were connected by SOE-PCR via linker sequence encoding five amino acids(G-G-G-G-S),the recombination expression plasmid pET-22b(+)-F6 was constructed and expressed in E.coli BL21.The expression proteinum was purified,and the blood serum was prepared by the immune cobaya,The specificity and sensitivity of antibody were verified by the ELISA and agar diffusion reaction.[Results] The fusion gene F6(HblA—VT1B—SEA—VT2B—BoNTaHc—SEB)and re-combinant plasmid pET-22b(+)-F6 was successfully constructed.The most of the 37℃ expression proteinum showed to be cyt-orrhyctes(expreesion amounted to 10.2%).The sequence encoding the mature fusion protein of the F6 toxin gene was 3384bp,encoding 1127 amino acids.The molecular weight of recombinant fusion toxin protein was 127.205 ku.The result of sequencing was consistent with predicted gene sequences.The results of ELISA and agar diffusion reaction demonstrated that fusion gene F6 and other four kinds of food poisoning-born bacteria had favorable reactionogenicity and did not response to many other non-tar-get bacteria.[Conclusion] The expression plasmid of multi-valent fusion toxin was successful constructed and antiserum was prepared,which laid the foundation for establishing broad spectrum and rapid detection method for food poisoning bacteria by utilization of fusion toxin detection methods.国家自然基金资助项目(30671762);厦门市科技计划项目(3502Z20055009
