3种中毒性弧菌融合毒素基因的表达与免疫学性质研究

Expression and Immunological Character Research of Three Kinds of Food-poisoning Vibrio Poly-recombinant Toxin Gene

为构建3种中毒性弧菌多联融合毒素基因及重组表达载体,制备多联融合毒素的血清抗体,本试验采用柔性Linker序列(Gly4Ser)对目的基因进行串联(tdh-vvhA-ctB),构建重组表达质粒pET-22b(+)-TVC并在原核表达载体内进行表达,将表达蛋白纯化后免疫动物制备多联融合毒素血清抗体,利用琼脂扩散试验和酶联免疫吸附试验验证抗体的特异性与敏感性。结果表明,试验成功构建了多联融合毒素重组表达质粒pET-22b(+)-TVC,并在原核表达载体内成功表达,表达量为11.38%,表达蛋白主要为包涵体,少量为可溶性蛋白,基因序列全长2 196bp,编码731个氨基酸,蛋白分子质量为81.7ku,测序结果与设计序列同源性为99.6%。ELISA和琼脂扩散试验表明,融合毒素TVC与3种目标中毒性弧菌均发生反应,与多种非目标菌均不反应。本试验成功构建了多联融合毒素基因的表达质粒并制备了抗血清,为利用重组毒素的方法检测目标毒素,进而建立更广谱的食物中毒菌快速检测方法奠定基础。

In this study,tdh-vvhA-ctB was constructed using the flexible Linker sequence（Gly4Ser）in order to construct three kinds of food-poisoning Vibrio poly-recombinant toxin gene and recombinant expression vector.The recombinant expression plasmid pET-22b（＋）-TVC was constructed and expressed in prokaryotic expression vector.The animals were immunized using the expressed protein after purification to get serum antibody.The specificity and sensitivity of the antibody were verified by agar diffusion test and enzyme-linked immunosorbent assay（ELISA）.The results showed that the recombinant expressing plasmid pET-22b（＋）-TVC was constructed and expressed successfully in prokaryotic expression vector,the expression level was11.38%.The expressed protein was mainly inclusion body and a small amount of soluble protein.The gene length was 2 196 bp,encoding 731 amino acids with molecular weight of 81.7ku.The results were 99.6% homologous to the designed sequence.Agar diffusion reaction and ELISA tests indicated the poly-recombinant toxin TVC could product different immune intersect reaction with other food-poisoning Vibrios but not react with some no-objective bacteria.Expression plasmid of poly-recombinant toxin gene was constructed and serum antibody was prepared successfully.It might be used to check the objective toxin based on the poly-recombinant toxin gene and established theboard-spectrum,quick,special detecting way to lay the theoretical and technical basis.