3种中毒性弧菌融合毒素基因的表达与免疫学性质研究

发布时间：2018-04-26 22:16

  本文选题：中毒性弧菌 + 多联融合毒素　； 参考：《中国畜牧兽医》2017年08期

【摘要】：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。
[Abstract]:In order to construct three vibrio vibrio vibrio multiplex fusion toxin genes and recombinant expression vectors to prepare the serum antibody of multiplex fusion toxin. In this experiment, a recombinant expression plasmid pET-22bVC was constructed and expressed in a prokaryotic expression vector by using the flexible Linker sequence Gly4Sera to construct the recombinant plasmid pET-22bVC. After purification of the expressed protein, the multiplex fusion toxin serum antibody was prepared by immunizing the target gene with tdh-vvhA-ctBnb, and the recombinant plasmid pET-22bVC was expressed in the prokaryotic expression vector. Agar diffusion test and enzyme-linked immunosorbent assay (Elisa) were used to verify the specificity and sensitivity of the antibody. The results showed that the recombinant expression plasmid pET-22b (pET-22b) was successfully constructed and expressed in prokaryotic expression vector (11.38%). The protein was mainly inclusion body and a small amount of soluble protein. The total length of the gene was 2196bp, encoding 731 amino acids, and the molecular weight of the protein was 81.7 ku.The homology between the sequence and the designed sequence was 99.6%.ELISA and Agar diffusion test, indicating that the fusion toxin TVC reacted with the three target vibrio. It did not react with many non-target bacteria. In this experiment, the expression plasmid of multiplex fusion toxin gene was successfully constructed and antiserum was prepared, which laid a foundation for the detection of target toxin by the method of recombinant toxin and the establishment of a wider spectrum rapid detection method for food poisoning bacteria.
【作者单位】： 唐山出入境检验检疫局;
【基金】：河北出入境检验检疫局科技项目(HE2014K034)
【分类号】：S941.4
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本文编号：1807918