發(fā)布時間：2018-04-26 22:16

  本文選題：中毒性弧菌 + 多聯(lián)融合毒素�。� 參考：《中國畜牧獸醫(yī)》2017年08期

【摘要】：為構(gòu)建3種中毒性弧菌多聯(lián)融合毒素基因及重組表達載體,制備多聯(lián)融合毒素的血清抗體,本試驗采用柔性Linker序列(Gly4Ser)對目的基因進行串聯(lián)(tdh-vvhA-ctB),構(gòu)建重組表達質(zhì)粒pET-22b(+)-TVC并在原核表達載體內(nèi)進行表達,將表達蛋白純化后免疫動物制備多聯(lián)融合毒素血清抗體,利用瓊脂擴散試驗和酶聯(lián)免疫吸附試驗驗證抗體的特異性與敏感性。結(jié)果表明,試驗成功構(gòu)建了多聯(lián)融合毒素重組表達質(zhì)粒pET-22b(+)-TVC,并在原核表達載體內(nèi)成功表達,表達量為11.38%,表達蛋白主要為包涵體,少量為可溶性蛋白,基因序列全長2 196bp,編碼731個氨基酸,蛋白分子質(zhì)量為81.7ku,測序結(jié)果與設(shè)計序列同源性為99.6%。ELISA和瓊脂擴散試驗表明,融合毒素TVC與3種目標(biāo)中毒性弧菌均發(fā)生反應(yīng),與多種非目標(biāo)菌均不反應(yīng)。本試驗成功構(gòu)建了多聯(lián)融合毒素基因的表達質(zhì)粒并制備了抗血清,為利用重組毒素的方法檢測目標(biāo)毒素,進而建立更廣譜的食物中毒菌快速檢測方法奠定基礎(chǔ)。
[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.
【作者單位】： 唐山出入境檢驗檢疫局;
【基金】：河北出入境檢驗檢疫局科技項目(HE2014K034)
【分類號】：S941.4
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本文編號：1807918