用双歧杆菌构建产肠毒素大肠杆菌LTB口服活疫苗及其粘膜免疫佐剂功能研究

发布时间：2018-05-07 11:05

本文选题：产肠毒素大肠杆菌(ETEC) + 双歧杆菌　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 在世界范围内,产肠毒素大肠杆菌(ETEC)感染是引起腹泻最重要的因素之一。它是发展中国家腹泻发病和婴儿死亡的主要原因,也是旅行者腹泻与国家士兵腹泻的最常见病原菌。因此,一个安全有效的抵抗ETEC腹泻的疫苗对公众健康是非常重要的。 ETEC是非侵袭性的,是依靠定居因子(CFA)粘附小肠粘膜上的,进而分泌肠毒素LT和ST。CFAs是细菌细胞表面的鞭毛,可以促进对小肠上皮细胞的粘附,主要有CFA/I、CFA/II和CFA/1V家族菌毛抗原。最普遍的CFAs是CFA/I。ST是由estA基因编码的19个氨基酸组成的单体多肽,免疫原性很差。LT是由eltAB编码,结构与霍乱毒素(CT)相似。它由两个亚单位组成即有毒性的A单位(LTA)和五聚体的B单位(LTB)组成。CT和LT有很强的免疫原性和粘膜佐剂活性。LTB也和CTB一样有很强的免疫原性和佐剂活性。本研究中选用LTB作为免疫原和粘膜免疫佐剂。 ETEC疫苗要求能够中和大多数ETEC菌株的毒力因子抗原,目前普遍认为一个合理的大肠杆菌疫苗应该包括三个主要的菌毛抗原即CFA/I,CFA/II,和CFA/IV以及具有免疫原性的不耐热肠毒素LT。因此在研究LTB免疫活性时选用CFA/I为参照。目前ETEC疫苗研究热点集中在三个方向:1.免疫方式由传统的肌肉、皮下等转为粘膜免疫;2.免疫途径上寻求粘膜佐剂,常用CT和LT;3.表达载体由传统的有毒转向减毒或无毒载体。根据以上三点,我们采用无毒的双歧杆菌为表达载体,通过粘膜免疫SD大鼠,检LTB的免疫原性,并与重组的双歧杆菌-CFA/I疫苗共免疫以检测其粘膜佐剂活性。双歧杆菌是人类肠道的自然宿主且可以粘附于肠道上皮细胞。因此,本研究的目的是将双歧杆菌发展成一个表达LTB蛋白的口服活疫苗的抗原表达系统。目的:构建携带ETEC LTB的双歧杆菌重组疫苗,然后将此载体疫苗免疫SD大鼠,检测其在大鼠体内诱导的体液和粘膜免疫应答,并与双歧杆菌重组的pBES-CFA/I疫苗共免疫,检测其粘膜免疫佐剂的效应。方法:(1)以pBV220为基础,构建穿梭表达载体pBES-LTB。将其电转化婴儿双歧杆菌,SDS-PAGE验证蛋白的表达,并通过家兔肠袢实验验证表达蛋白的安全性。(2)用双歧杆菌重组载体疫苗免疫SD大鼠:随机分四组分别为PBS、pBES-LTB、pBES-CFA/I和pBES-LTB+PBES-CFA/I组,每组12只,免疫三次(0,10,17天),并于0,7,10,14,17,22和27天采血和粪便样本,ELISA检测其特异抗体水平。(3)在第27天,每组一半大鼠腹腔感染致死剂量ETEC毒株H10407,连续观察20天,计算其存活力。另一半鼻饲ETEC H10407,观察其肺部感染情况。结果:(1)LTB蛋白在双歧杆菌中成功表达,其表达蛋白经家兔肠袢实验证实是微毒的。(2)ELISA结果表明:pBES-LTB与pBES-CFA/I疫苗联合免疫组的大鼠比其它单独免疫组产生了更强烈的血清IgG和粪便IgA抗体(P0.05)。LTB免疫组和CFA/I免疫组间差别无统计学意义(P0.05)。(3)腹腔攻毒保护实验结果证实, pBES-LTB + pBES-CFA/I免疫组的SD大鼠保护性比单独免疫组的好,单独口服天然双歧杆菌和PBS的免疫组无保护性。(4)ETEC鼻饲实验结果表明:pBES-LTB +pBES-CFA/I免疫组及pBES-CFA/I单独免疫的SD大鼠肺部均无病理变化, pBES-LTB免疫组有一定的炎症反应,未免疫组肺部有严重的病理变化。结论:(1)双歧杆菌可以作为ETEC重组口服活疫苗的表达载体系统,该口服疫苗表达系统开辟了ETEC疫苗研究的新方向;(2)双歧杆菌表达的LTB单独接种对ETEC的粘附无免疫保护性,但肠袢试验证明它对LT具有免疫性;(3)pBES-LTB与pBES-CFA/I联合免疫,可明显提高CFA/I的抗体滴度,并使动物获得更好的保护力,即具备口服疫苗免疫佐剂的基本特性。
[Abstract]:Enterotoxigenic Escherichia coli (ETEC) infection is one of the most important causes of diarrhoea in the world. It is the main cause of diarrhoea and infant death in developing countries. It is also the most common pathogen of traveller diarrhoea and national soldiers' diarrhea. Therefore, a safe and effective vaccine against ETEC diarrhea is not for public health. It's often important.
ETEC is non invasive and relies on the colonization factor (CFA) adhering to the small intestinal mucosa, and then secreting enterotoxin LT and ST.CFAs is the flagellum on the surface of the bacterial cells, which can promote the adhesion to the epithelial cells of the small intestine, mainly CFA/I, CFA/II and CFA/1V family pili antigens. The most common CFAs is that CFA/I.ST is a 19 amino acid group encoded by estA genes. .LT, a poor immunogenicity, is encoded by eltAB. The structure is similar to cholera toxin (CT). It consists of two subunits consisting of toxic A units (LTA) and B units (LTB) composed of.CT and LT, which have strong immunogenicity and mucous adjuvant activity, which have strong immunogenicity and adjuvant activity as.LTB and CTB. LTB was used as immunogen and mucosal immune adjuvant.
ETEC vaccine requires the ability to neutralize the virulence factor antigens of most ETEC strains. It is widely believed that a reasonable Escherichia coli vaccine should include three major hair antigens, namely, CFA/I, CFA/II, CFA/IV, and immunogenic non thermal enterotoxin LT.. Therefore, the use of CFA/I as a reference for the study of LTB immunological activity. The hot spots in the study of seedlings are concentrated in three directions: 1. the immune mode is transformed from the traditional muscle and subcutaneous into the mucosal immunity, and the 2. immune pathway seeks the mucosal adjuvant, commonly used CT and LT; the 3. expression vector is from the traditional toxic turn reducing or nontoxic carrier. According to the above three points, we use the non toxic Bifidobacterium as the expression vector and immunization SD through mucous membrane. The immunogenicity of LTB was detected and co immunized with recombinant Bifidobacterium -CFA/I vaccine was used to detect its mucosal adjuvant activity.
Bifidobacterium is a natural host of human intestinal tract and can adhere to intestinal epithelial cells. Therefore, the aim of this study is to develop the Bifidobacterium into an oral live vaccine expressing LTB protein for the antigen expression system.
Objective: to construct a recombinant vaccine of Bifidobacterium carrying ETEC LTB, and then immunization the SD rats with this vaccine to detect the humoral and mucosal immune responses in rats, and co immunization with the recombinant pBES-CFA/I vaccine of bifidobacteria to detect the effect of its mucosal immune adjuvant.
Methods: (1) on the basis of pBV220, the shuttle expression vector pBES-LTB. was constructed to convert the electric conversion of Bifidobacterium and SDS-PAGE to the expression of protein, and the safety of the expression protein was verified by the rabbit intestinal loop experiment. (2) SD rats were immunized with Bifidobacterium recombinant vector vaccine: four groups were randomly divided into four groups: PBS, pBES-LTB, pBES-CFA/I and pBES-LTB+PBES-, respectively. Group CFA/I, 12 rats in each group, immunized three times (0,10,17 days), and samples of blood collection and feces on 0,7,10,14,17,22 and 27 days, and ELISA detection of specific antibody levels. (3) in the twenty-seventh day, the lethal dose of ETEC strain H10407 in the abdominal infection of half of each group of rats in each group was observed for 20 days and its viability was calculated. The other half of the nasal feeding was ETEC H10407, and the pulmonary infection was observed.
Results: (1) LTB protein was successfully expressed in bifidobacteria, and its expression protein was proved to be micro toxic by rabbit intestinal loop experiment. (2) ELISA results showed that the rats of pBES-LTB and pBES-CFA/I vaccine combined immunization group produced more intense serum IgG and fecal IgA antibody (P0.05).LTB immune group and CFA/I immune group than other immune groups. Statistical significance (P0.05) (3) the experimental results of intraperitoneal attack protection confirmed that the SD rats in the pBES-LTB + pBES-CFA/I immunization group were better than those of the single immune group. (4) the experimental results of ETEC nasal feeding showed that the pBES-LTB +pBES-CFA/I immune group and the SD rats of pBES-CFA/I alone were immune. No pathological changes were found in the lungs. There was a certain inflammatory reaction in the pBES-LTB immuno group, and severe pathological changes in the lungs of the unimmunized group.
Conclusions: (1) bifidobacteria can be used as an expression vector system for ETEC recombinant oral live vaccine. The oral vaccine expression system opens a new direction for the study of ETEC vaccine. (2) the adhesion of LTB expressed by Bifidobacterium is not immune to ETEC, but the intestinal loop test shows that it has immunity to LT; (3) pBES-LTB and pBES-CFA/I are immune to immunization. Epidemics can significantly increase the antibody titer of CFA/I and provide better protection for animals, that is, the basic characteristics of oral adjuvants.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【相似文献】