用双歧杆菌构建产肠毒素大肠杆菌定居因子I重组载体疫苗研究

发布时间：2018-03-12 07:37

本文选题：产肠毒素型大肠杆菌(ETEC)　切入点：双歧杆菌　出处：《重庆医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 大肠杆菌感染是引起腹泻最重要的因素之一。在许多发展中国家,肠毒素大肠杆菌ETEC是导致婴幼儿腹泻致严重脱水的第二大病原菌(仅位于轮状病毒之后),ETEC也是旅行者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。现已知ETEC的两种抗原—定居因子抗原(colonization factor antigens,CFAs)和肠毒素(enterotoxins)参与了腹泻过程。ETEC菌体表面的菌毛(又称定居因子抗原)是重要的致病因子,ETEC感染宿主后,正是靠定居因子粘附干宿主小肠上皮细胞,经定居、繁殖产生肠毒素而致病。迄今为止,了解得比较清楚的菌毛抗原包括定居因子抗原I (CFA/I)、定居因子抗原II (CFA/ II)和定居因子抗原IV (CFA/ IV) [1]。其中CFA/I是一种优势血清型定居因子,其相应抗体在阻止病原体在宿主小肠定居而引起腹泻方面起重要怍用,在许多地方流行腹泻区,它是许多疫苗的一个重要组成成分。肠毒素是ETEC产生的毒性分泌蛋白,有不耐热肠毒素(heat—labile entemtoxin,LT)和耐热肠毒素(heat—stable entemtoxin,sT)两种。CT和LT有很强的免疫原性和佐剂活性。LTB为无毒性的LT的B亚单位,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。目前ETEC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.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。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CFA/I的口服活疫苗的抗原表达系统。目的:构建携带ETEC CFA/I的双歧杆菌重组疫苗(即pBEX-CFA/I),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LTB(即pBES-LTB)共免疫,检测其粘膜免疫佐剂的效应。方法:(1)以pGEX-5x-1为基础,构建穿梭表达载体pBEX-CFA/I。将其电转化婴儿双歧杆菌,SDS-PAGE验证蛋白的表达。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、pBEX-CFA/I和pBES-LTB+pBEX-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ETECH10407,观察其肺部感染情况。结果:(1)CFA/I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无毒的。(2)ELISA结果表明:pBEX-CFA/I与pBES- LTB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)。CFA/I免疫组和LTB免疫组间无显著性差别(P0.05)。(3)腹腔攻毒保护实验结果证实, pBEX-CFA/I + pBES-LTB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的未免疫组无保护性。(4)ETEC鼻饲实验结果表明:pBEX-CFA/I+pBES-LTB免疫组及pBEX-CFA/I单独免疫的SD大鼠肺部均无病理变化。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)重组双歧杆菌表达-CFA/I单独接种对ETEC的粘附和感染有免疫保护作用;(3)双歧杆菌-CFA/I和双歧杆菌-LTB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。
[Abstract]:Escherichia coli infection is one of the most important factors of diarrhea. In many developing countries, enterotoxigenic Escherichia coli ETEC is the second leading pathogen of infantile diarrhea caused by severe dehydration (only after rotavirus), the most common pathogens of ETEC and traveler's diarrhea. Therefore, a safe and effective vaccine against ETEC is very important to public health.
It is known that the two ETEC antigen - colonization factor antigen (colonization factor, antigens, CFAs) and enterotoxin (enterotoxins) involved in the process of diarrhea.ETEC cell surface fimbriae (also called colonization factor antigen) is an important pathogenic factor, ETEC infection of the host, it is by colonization factor host intestinal epithelial cell adhesion stem, through the settlement breeding produce enterotoxin and disease., so far, to understand more clearly including fimbrial antigen I colonization factor antigen (CFA/I), II (CFA/ II colonization factor antigen) and IV (CFA/ colonization factor antigen [1]. CFA/I IV) which is a dominant serotype colonization factor, the corresponding antibody in preventing the pathogen in the host small intestine diarrhea caused by settlement plays an important effect in many places, epidemic diarrhea area, it is an important component of many vaccines. The toxicity of ETEC enterotoxin is a secreted protein, is not heat Enterotoxin (heat - labile entemtoxin, LT) and heat labile enterotoxin (heat - stable entemtoxin, sT) two.CT and LT B subunit strong immunogenicity and adjuvant activity of.LTB is non-toxic LT, also CTB and have strong immunogenicity and adjuvant activity in this study. Choose LTB as a mucosal adjuvant.
ETEC vaccine can request virulence factors or antigens of most ETEC strains, is considered a reasonable Escherichia coli vaccine should include three major fimbrial antigen CFA/I, CFA/II and CFA/IV, and has the immunogenicity of heat labile toxin LT. ETEC hotspot study focused on three aspects: 1. from the traditional way of immunity the muscle, subcutaneous tissue to mucosal immunity; 2. for mucosal adjuvant, commonly used CT and LT; 3. expression vector by toxic to the traditional attenuated or non-toxic carrier.
Bifidobacterium is a natural host of human intestinal tract and can adhere to intestinal epithelial cells. Therefore, the purpose of this study is to develop bifidobacteria into an antigen expression system for oral live vaccine expressing CFA/I.
Objective: to construct ETEC recombinant vaccine Bifidobacterium CFA/I (pBEX-CFA/I), and then the vector vaccine SD rats were detected in rats induced by in vivo humoral and mucosal immune responses, and Bifidobacterium recombinant LTB (pBES-LTB) were immunized, detection effect of the mucosal immune adjuvant.
Methods: (1) on the basis of pGEX-5x-1, construct the shuttle expression vector pBEX-CFA/I. transformed b.infantis, expression of SDS-PAGE protein. The expression of safety verification protein by rabbit intestinal loop experiments. (2) with recombinant vector vaccine Bifidobacterium SD rats were randomly divided into four groups: PBS, pBES-LTB pBEX-CFA/I, and pBES-LTB+pBEX-CFA/I group, 12 rats in each group, three times of immunization (0,10,17 days), and in 27 days 0,7,10,14,17,22 and blood and fecal samples, ELISA to detect the specific antibody level. (3) on the twenty-seventh day, half of each group of rats with abdominal infection a lethal dose of ETEC strain H10407, continuous observation for 20 days, calculate its viability the other half feeding ETECH10407, observe the pulmonary infection.
Results: (1) the successful expression of CFA/I protein in Bifidobacterium and its protein expression by the rabbit intestinal loop experiment proved to be non-toxic. (2) the result of ELISA showed that pBEX-CFA/I and pBES- LTB vaccine combined with immune group rat serum IgG and fecal IgA antibody was more strongly than other individual immune group (P0.05).CFA/I immune group and LTB immune group had no significant difference (P0.05). (3) intraperitoneal challenge protection experiment results proved that the protection of SD rats pBEX-CFA/I + pBES-LTB immune group than in immune group, is disease group alone oral Bifidobacterium and natural PBS without protection (4). The experimental results show that ETEC nasal pBEX-CFA/I+pBES-LTB immune group and pBEX-CFA/I alone immune SD rat lungs showed no pathological changes.
Conclusion: (1) Bifidobacterium ETEC can be used as a recombinant oral live vector vaccine expression system, the oral vaccine expression system opens up a new direction for the study of ETEC vaccine; (2) recombinant Bifidobacterium -CFA/I single inoculation on ETEC expression of adhesion and infection immunity; (3) Bifidobacterium -CFA/I and Bifidobacterium -LTB combined immunization can significantly improve the antibody titer of CFA/I, and make the animal protection better.

【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

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