食源性致病菌六联融合毒素的构建及免疫学特性研究  

STUDY ON THE CONSTRUCTION AND IMMUNOLOGY CHARACTERISITCS OF SIX-VA-LENT FUSION TOXIN OF FOOD POISONING-BORNE BACTERIA

作　　者：宫彬彬[1] 柳增善[1] 于师宇[1] 孟究梅[1] 卢士英[1] 任洪林[1,2] 李兆辉[1] 王克坚[2] 

机构地区：[1]吉林大学人兽共患病研究所,人兽共患病教育部重点实验室,长春130062 [2]近海海洋环境科学国家重点实验室,厦门大学海洋与环境学院

出　　处：《现代预防医学》2009年第6期1122-1126,共5页Modern Preventive Medicine

基　　金：国家自然基金资助项目(30671762);厦门市科技计划项目(3502Z20055009)

摘　　要：[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 （ HbIA 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. colt BL21. The expression proteinum was purified, and the blood serum was prepared by tile immune eobava. The specificity and sensitivity of antibody were verified by the ELISA and agar diffiusion reaction. [Results] The fusion gene F6 （HbIA--VT1B--SEA--VT2B--BoNTaHc SER） and recombinant plasmid pET-22b（＋）-F6 was successfltllv constructed. The most of the 37℃ expression proteinum showed to be cytorrthyctes （expreesion amounted to 10.2%）. The sequence encoding the mature filsion 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 reaetionogenielty and did not response to many other non-target bacteria. [ Conclusion] The expression plasmid of muhi-vaJcnt 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.

关 键 词：食源性致病菌 基因串联 融合毒素 血清抗体 ELISA检测 

分 类 号：R392[医药卫生—免疫学]