發(fā)布時(shí)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(guó)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(dá)載體,制備多聯(lián)融合毒素的血清抗體,本試驗(yàn)采用柔性L(fǎng)inker序列(Gly4Ser)對(duì)目的基因進(jìn)行串聯(lián)(tdh-vvhA-ctB),構(gòu)建重組表達(dá)質(zhì)粒pET-22b(+)-TVC并在原核表達(dá)載體內(nèi)進(jìn)行表達(dá),將表達(dá)蛋白純化后免疫動(dòng)物制備多聯(lián)融合毒素血清抗體,利用瓊脂擴(kuò)散試驗(yàn)和酶聯(lián)免疫吸附試驗(yàn)驗(yàn)證抗體的特異性與敏感性。結(jié)果表明,試驗(yàn)成功構(gòu)建了多聯(lián)融合毒素重組表達(dá)質(zhì)粒pET-22b(+)-TVC,并在原核表達(dá)載體內(nèi)成功表達(dá),表達(dá)量為11.38%,表達(dá)蛋白主要為包涵體,少量為可溶性蛋白,基因序列全長(zhǎng)2 196bp,編碼731個(gè)氨基酸,蛋白分子質(zhì)量為81.7ku,測(cè)序結(jié)果與設(shè)計(jì)序列同源性為99.6%。ELISA和瓊脂擴(kuò)散試驗(yàn)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(yàn)成功構(gòu)建了多聯(lián)融合毒素基因的表達(dá)質(zhì)粒并制備了抗血清,為利用重組毒素的方法檢測(cè)目標(biāo)毒素,進(jìn)而建立更廣譜的食物中毒菌快速檢測(cè)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(yàn)檢疫局;
【基金】：河北出入境檢驗(yàn)檢疫局科技項(xiàng)目(HE2014K034)
【分類(lèi)號(hào)】：S941.4
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本文編號(hào)：1807918