食物中毒菌多联融合毒素Hc-VT1B-SEA-VT2B-SEB的表达及免疫学特性研究

Expression of the multi-valent fusion toxin of food poisoning-borne bacteria and its immulogic properties

目的构建食物中毒菌多联融合毒素基因及重组表达载体,制备抗5种毒素的血清抗体。方法采用一定的linker序列(G-P-G-P-G)将克隆的3种食物中毒菌的5个毒素基因片段进行串联,并定向克隆到表达载体pET-22b(+)的NcoI和XhoI位点之间,构建五联融合毒素基因及重组表达载体,表达产物经切胶回收初步纯化后免疫獭兔,制备抗血清,利用ELISA和琼脂扩散试验检测抗体和5种毒素反应的特异性和敏感性。结果构建了重组融合毒素(命名为F5)Hc-VT1B-SEA-VT2B-SEB基因和重组表达载体F5-22b,且在表达宿主菌E.coliBL21中得到有效表达(9.90%),基因序列全长2937bp,编码成熟蛋白979个氨基酸,融合蛋白分子量112.369ku,测序结果与设计序列(GenBank)100%同源。表达产物经初步纯化后免疫獭兔获得抗五种毒素的血清抗体,且具有较高的特异性和敏感性。结论成功构建了融合毒素基因,获得了抗五种毒素的血清抗体,本实验结果为下一步建立食品广谱、快速免疫学检测新方法奠定了基础。

Five gene fragments from E.coli O157∶H7,Staphylococcus aureus and C.botulinum were connected by SOE PCR via linker sequence encoding five amino acids（G-P-G-P-G）.After digested with restriction nuclease NcoI and XhoI,the linked gene was cloned into pMD18-T vector.The resultant recombinant plasmid,termed F5,was transformed into E.coli DH5α and then linked into expression vector pET-22b（＋）,termed F5-22b,was transformed into host strain E.coli BL21（DE3）.The recombinant toxin expressed was induced by IPTG and identified by SDS-PAGE.The expressed product was immuned rabbits to obtain serum antibody.In this way fusion gene F5（Hc-VT1B-SEA-VT2B-SEB）and recombinant plasmid F5-22b were constructed.The sequenced results of the fusion gene F5 was consistent with the predicted gene sequences.The sequence encoding the mature fusion protein of the F5 toxin gene was 2 937bp,encoding 979 amino acids.The molecular weight of recombinant fusion toxin protein was 112.369 ku.The expressed protein amounted to 9.90% of the total protein of E.coli BL21（DE3）after induced by IPTG（1mmol/L）for 4 hours.Two expressed products including soluble protein in pET-22b and inclusion body were obtained.Serum antibodies against five toxins were obtained from rabbits immunized with expressed products.ELISA and agar diffusion assay showed that the antibodies had high reactive specificity and sensitivity with five toxins.The findings of this experiment would lay foundations for the establishment of the broad-spectrum immunologic methods to detect food poisoning borne bacteria or toxins.