【作者】 黄思扬；

【导师】 王锐； 沈心亮；

【作者基本信息】 兰州大学 ， 生物学化学与分子生物学， 2006， 硕士

【摘要】 目前所知道的最强的粘膜免疫抗原当属霍乱毒素(cholera toxin CT)与大肠杆菌不耐热肠毒素(heat-labile enterotoxin,LT),但是两者分别能引起典型的霍乱样腹泻,及旅行者腹泻。同时它具有很强的潜在的免疫原性及粘膜免疫佐剂效应,以他们作为佐剂能够显著刺激增强机体对抗原的免疫反应,但是其毒性限制了其的应用,随着LT的三维结构的获悉,推动了人们对其B亚单位及其减毒突变体的研究,期望得到具有很强的粘膜佐剂活性,同时其毒性具有相对较低甚至没有毒性的突变体。 本实验的目的是为了研究大肠杆菌不耐热肠毒素(Escherichia coli heat-labile enterotoxin(LT))B亚单位(LTB)的免疫佐剂活性。从产肠毒素大肠杆菌44815#菌株中提取其大质粒DNA,根据GENE BANK的序列设计引物调出LTB的原始基因,并将该基因通过PCR的手段进行扩增。进而将该基因克隆入pMD 18-T vector,通过PCR及双酶切进行筛选鉴定,阳性克隆测序。将正确的基因克隆到原核表达载体pET-21b(+)中,得到重组原核表达株(pET21b-LTB),经氯化钙法转化大肠杆菌BL21(DE3),通过PCR及IPTG诱导表达经过SDS-PAGE筛选阳性克隆,命名为pET21b-LTB。表达产物经SDS-PAGE分析,分子量为14KD,与理论值相符。凝胶灰度扫描显示pET21b-LTB表达量约占菌体总蛋白的16%。 将构建好的重组菌株进行扩大培养,离心收集细胞,经超声破碎后离心收集破碎上清、通过阳离子交换柱(CM-FF)对目的蛋白进行初步纯化。以该纯化蛋白为佐剂,将其分别与流感三价疫苗,Ⅱ型疱疹病毒(HSV-Ⅱ)gD糖蛋白混合后通过腹腔注射及鼻饲法免疫BALB/c小鼠,设单纯铝佐剂免疫为对照。免疫2,4,6周后尾静脉采血,ELISA法检测其抗体水平,检测抗体水平升高后。将小鼠摘除眼球采血,全部处死后取其鼻、肺、肠及阴道洗液检测sIgA抗体水平,取其血清检测IgA、IgM、IgG抗体水平。通过与氢氧化铝佐剂对比表明其滴度显著高于氢氧化铝佐剂对照组。 结果表明:重组大肠杆菌不耐热肠毒素B亚单位具有粘膜免疫佐剂功效,该研究结果为今后LTB的研究及应用奠定了基础。更多还原

【Abstract】 The most powerful mucosal immunogens recognized to date are cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT), the molecules that cause the debilitating watery secretions typical of cholera and traveller’s diarrhoea, respectively . The anti-toxin response induced is so potent that a strong immune response is also activated against foreign bystander molecules that are present simultaneously at the mucosal surface. Nevertheless, the use of LT as mucosal immunogens and adjuvants in humans has been limited by their toxicity. The three-dimensional structure of LT has been solved and these results have prompted attempts to dissect, using genetic techniques, the mucosal immunogenicity and adjuvant activity of LT from their toxicity. Such studies have allowed to design non toxic but immunogenic molecules and to define the roles of the receptor-binding B domain, the A subunit, and the enzymatic activity of LT in mucosal immunogenicity and adjuvanticity.To study the immunoadjuvanticity of recombinant Escherichia coli heat -labile enterotoxin B subunit (rLTB), we expressed LTB in E.coli and tested the antibody that generated after immune, by influent vaccine or HSV-II together with LTB.Firstly, we amplified the LTB·gene from large plasmid that extracted from ETEC 44815# strain by PCR. Then, we cloned this original PCR product into pMD18-T vector for sequencing, whose result showed that the gene wecloned was exactly LTB. Secondly, we tried to use pET21b(+) vector to express LTB in Escherichia coli BL2KDE3). Positive colony was confirmed by PCR screen and expression test. LTB protein was successfully expressed and its molecular weight on the gel of SDS-PAGE is 14KD which is consistent with the theoretical value.To test the immunoadjuvanticity of LTB, we performed large scale purification of this protein and used it as immunoadjuvant to immune mice together with influenza virus vaccine and HSV-II (gD) antigen respectively, which was controlled by without LTB and with Al(0H)3 as the adjuvant. BalB/C mice were administered via inject o or nasal rout. Collection of mucosal fluids, Nasal, lung, and gut washes were collected 3 days after the immunization. Nasal, lung, and gut lavages were performed on the sacrificed animal by repeated flushing by phosphate buffered saiine (PBS) containing 0.1% bovine serum albumin (BSA). Serum samples and vaginal fluids were collected 3 days after the last immunization.We use ELISA to test the antibody that generated after the immunity and killed the animal to test slgA from nose, lung, intestines and vagina or IgG, IgA, IgM from serum. The results demonstrated that LTB is an effective mucosal immunoadjuvant and its adjuvant activity is stronger than the control.更多还原