【摘要】： 產(chǎn)腸毒素大腸桿菌(Enterotoxic Escherichia coli)是一類致人和幼畜腹瀉的最常見的致病性大腸桿菌,初生幼畜感染后常見因劇烈水樣腹瀉和迅速脫水而死亡,發(fā)病率和病死率均很高。大腸桿菌不耐熱腸毒素(heat-labile enterotoxin,LT)是產(chǎn)腸毒素大腸桿菌分泌的一種不耐熱腸毒素,是引起人和家畜腹瀉的大腸桿菌的致病因子。LT是由一個(gè)具有ADP-核糖基轉(zhuǎn)移酶活性的A亞基和具有GM1-神經(jīng)節(jié)苷脂受體結(jié)合位點(diǎn)的同源五聚體B亞基組成的六聚蛋白。盡管LT有很強(qiáng)的毒性,但其同時(shí)具有很強(qiáng)的免疫原性并且能夠輔佐其他抗原通過粘膜免疫途徑使機(jī)體產(chǎn)生特異抗體,因而LT在粘膜免疫研究中以及粘膜免疫疫苗開發(fā)中具有重要醫(yī)學(xué)價(jià)值和經(jīng)濟(jì)價(jià)值。 為構(gòu)建無(wú)毒又保持粘膜免疫佐劑活性的LT突變體,進(jìn)一步研究LT的免疫機(jī)理及其在基因工程疫苗研制上的應(yīng)用,本研究根據(jù)毒素的結(jié)構(gòu)及作用機(jī)理,通過基因工程的方法以不耐熱腸毒素44814株為材料,應(yīng)用PCR重疊延伸法對(duì)大腸桿菌不耐熱腸毒素A亞單位進(jìn)行擴(kuò)增和修飾,將第63位編碼絲氨酸密碼子突變?yōu)榫幋a賴氨酸的密碼子,并用質(zhì)粒載體pGEX-6p-1在大腸桿菌BL21中進(jìn)行克隆及融合表達(dá),SDS-PAGE及Western-blot的結(jié)果分析表明獲得了LT突變體。 動(dòng)物實(shí)驗(yàn)將80羽1日齡非免疫健康肉雛雞隨機(jī)平均分成4組,Ⅰ、Ⅱ組在接種新城疫病毒LaSota株弱毒活疫苗的同時(shí),將大腸桿菌不耐熱腸毒素A亞單位基因突變株表達(dá)產(chǎn)物作為免疫佐劑按不同劑量口服,Ⅲ組僅免疫并用生理鹽水代替突變株表達(dá)產(chǎn)物,Ⅳ組為對(duì)照組,既不免疫也不口服突變株表達(dá)產(chǎn)物或生理鹽水。在免疫后的第7d、14d、21d、28d,分別測(cè)定各組動(dòng)物的抗NDV血凝抑制抗體和粘膜抗體水平。結(jié)果表明,突變體對(duì)提高雞體抗NDV血凝抑制抗體效價(jià)和粘膜抗體水平均具有一定作用。 同時(shí),本研究還利用初步提純的B亞單位的融合表達(dá)產(chǎn)物作為免疫原免疫6周齡BALB/c小鼠,取其脾細(xì)胞與SP2/0骨髓瘤細(xì)胞融合,用間接ELISA檢測(cè),經(jīng)三次亞克隆得到了穩(wěn)定分泌抗LTB單克隆抗體的雜交瘤細(xì)胞株G5和F7,并制備了腹水,利用該單抗初步建立了間接ELISA特異性檢測(cè)LT抗原的方法。為進(jìn)一步建立特異性強(qiáng)、敏感性高、簡(jiǎn)便快速的LT檢測(cè)方法奠定了基礎(chǔ)。
[Abstract]:Enterotoxigenic Escherichia coli (Enterotoxic Escherichia coli) is the most common pathogenic Escherichia coli causing diarrhea in human and young animals. E. coli heat-labile enterotoxin (heat-labile enterotoxin,LT) is a kind of heat-labile enterotoxin secreted by enterotoxigenic Escherichia coli. LT is composed of a subunit A with the activity of ADP- ribosyltransferase and a homologous pentamer B subunit with GM1- ganglioside receptor binding site. Although LT is highly toxic, it also has strong immunogenicity and adjusts other antigens to produce specific antibodies through mucosal immunity. Therefore, LT has important medical value and economic value in mucosal immunity research and mucosal immunization vaccine development. In order to construct nontoxic LT mutants with mucosal immune adjuvant activity, and to further study the immune mechanism of LT and its application in the development of genetically engineered vaccine, the present study was based on the structure and mechanism of the toxin. By genetic engineering, 44814 strains of heat-labile enterotoxin were used to amplify and modify Escherichia coli heat-labile enterotoxin A subunit by PCR overlapping extension method. The 63rd position encoding serine codon was mutated into the lysine codon. The plasmid vector pGEX-6p-1 was cloned and expressed in Escherichia coli BL21. The results of SDS-PAGE and Western-blot showed that the LT mutants were obtained. In animal experiments, 80 1-day-old unimmunized broiler chicks were randomly divided into 4 groups. Groups 鈪，

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