發(fā)布時(shí)間：2018-06-27 03:17

  本文選題：沙門菌鞭毛 + 基因重組��； 參考：《吉林大學(xué)》2008年碩士論文

【摘要】： 強(qiáng)致病性的甲型副傷寒沙門菌、鼠傷寒沙門菌和豬霍亂沙門菌Ⅰ相鞭毛抗原的血清型分別為a、i、c。針對(duì)這三種沙門菌Ⅰ相鞭毛抗原H1-a、H1-i、H1-c的編碼基因設(shè)計(jì)三對(duì)擴(kuò)增引物。首先,利用常規(guī)PCR技術(shù)擴(kuò)增出三段Ⅰ相鞭毛抗原基因,再利用引物上的Linker和重疊延伸PCR擴(kuò)增技術(shù)將此三段抗原決定區(qū)基因序列串聯(lián),獲得鞭毛重組基因。構(gòu)建鞭毛重組基因的原核表達(dá)載體pET-28a-F,并將重組表達(dá)質(zhì)粒轉(zhuǎn)化大腸桿菌BL21株。表達(dá)菌在終濃度為1mmol/L的IPTG,37℃下誘導(dǎo)6h后,SDS-PAGE電泳分析得到鞭毛融合蛋白大小約為95Ku,表達(dá)量約為11.2%。含有目的蛋白的包涵體經(jīng)初步純化后,免疫豚鼠獲得血清抗體。瓊脂擴(kuò)散試驗(yàn)和間接ELISA測(cè)定抗體效價(jià)可達(dá)到1:512000,此時(shí)抗體的敏感性為8μg/mL;血清抗體稀釋10000×?xí)r對(duì)甲型副傷寒沙門菌、鼠傷寒沙門菌、豬霍亂沙門菌檢出值分別為:1.35×10~5個(gè)/mL、1.63×10~5個(gè)/mL、2.31×105個(gè)/mL;用10種沙門菌和15種非沙門菌屬的其他食物中毒菌做免疫交叉反應(yīng),結(jié)果顯示特異性較好,同時(shí)抗體顯示很好的廣譜性,即能和相同鞭毛血清型的沙門菌菌群反應(yīng)。純化血清抗體并進(jìn)行膠體金免疫層析實(shí)驗(yàn),對(duì)試紙條檢測(cè)方法做了初步研究,為食品中沙門菌的快速檢測(cè)技術(shù)奠定基礎(chǔ)。
[Abstract]:The serotypes of strongly pathogenic Salmonella paratyphoid A, Salmonella typhimurium in mice and Salmonella cholerae in swine in the first phase of flagellum antigen were as follows. Three pairs of amplified primers were designed for the coding genes of the flagellum antigen H1-aH1-iH1-c of these three Salmonella species. Firstly, the three-segment I flagellum antigen gene was amplified by conventional PCR technique, and then the gene sequence of the three antigen-determining region was sequenced by Linker and overlapping extension PCR on the primer to obtain the flagellum recombinant gene. The prokaryotic expression vector pET-28a-Fof flagellum recombinant gene was constructed and transformed into Escherichia coli BL21 strain. The expression strain was induced at 37 鈩，

本文編號(hào)：2072461