大肠杆菌不耐热肠毒素突变体的构建及其粘膜免疫剂活性的研究与B亚单位单克隆抗体的制备
发布时间：2019-02-16 11:04
【摘要】： 产肠毒素大肠杆菌(Enterotoxic Escherichia coli)是一类致人和幼畜腹泻的最常见的致病性大肠杆菌,初生幼畜感染后常见因剧烈水样腹泻和迅速脱水而死亡,发病率和病死率均很高。大肠杆菌不耐热肠毒素(heat-labile enterotoxin,LT)是产肠毒素大肠杆菌分泌的一种不耐热肠毒素,是引起人和家畜腹泻的大肠杆菌的致病因子。LT是由一个具有ADP-核糖基转移酶活性的A亚基和具有GM1-神经节苷脂受体结合位点的同源五聚体B亚基组成的六聚蛋白。尽管LT有很强的毒性,但其同时具有很强的免疫原性并且能够辅佐其他抗原通过粘膜免疫途径使机体产生特异抗体,因而LT在粘膜免疫研究中以及粘膜免疫疫苗开发中具有重要医学价值和经济价值。 为构建无毒又保持粘膜免疫佐剂活性的LT突变体,进一步研究LT的免疫机理及其在基因工程疫苗研制上的应用,本研究根据毒素的结构及作用机理,通过基因工程的方法以不耐热肠毒素44814株为材料,应用PCR重叠延伸法对大肠杆菌不耐热肠毒素A亚单位进行扩增和修饰,将第63位编码丝氨酸密码子突变为编码赖氨酸的密码子,并用质粒载体pGEX-6p-1在大肠杆菌BL21中进行克隆及融合表达,SDS-PAGE及Western-blot的结果分析表明获得了LT突变体。 动物实验将80羽1日龄非免疫健康肉雏鸡随机平均分成4组,Ⅰ、Ⅱ组在接种新城疫病毒LaSota株弱毒活疫苗的同时,将大肠杆菌不耐热肠毒素A亚单位基因突变株表达产物作为免疫佐剂按不同剂量口服,Ⅲ组仅免疫并用生理盐水代替突变株表达产物,Ⅳ组为对照组,既不免疫也不口服突变株表达产物或生理盐水。在免疫后的第7d、14d、21d、28d,分别测定各组动物的抗NDV血凝抑制抗体和粘膜抗体水平。结果表明,突变体对提高鸡体抗NDV血凝抑制抗体效价和粘膜抗体水平均具有一定作用。 同时,本研究还利用初步提纯的B亚单位的融合表达产物作为免疫原免疫6周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA检测,经三次亚克隆得到了稳定分泌抗LTB单克隆抗体的杂交瘤细胞株G5和F7,并制备了腹水,利用该单抗初步建立了间接ELISA特异性检测LT抗原的方法。为进一步建立特异性强、敏感性高、简便快速的LT检测方法奠定了基础。
[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，
本文编号：2424377
[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，
本文编号：2424377
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