本文選題：產(chǎn)腸毒素型大腸桿菌(ETEC)　切入點(diǎn)：雙歧桿菌　出處：《重慶醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 大腸桿菌感染是引起腹瀉最重要的因素之一。在許多發(fā)展中國家,腸毒素大腸桿菌ETEC是導(dǎo)致嬰幼兒腹瀉致嚴(yán)重脫水的第二大病原菌(僅位于輪狀病毒之后),ETEC也是旅行者腹瀉的最常見病原菌。因此,一個安全有效的抵抗ETEC腹瀉的疫苗對公眾健康是非常重要的。 現(xiàn)已知ETEC的兩種抗原—定居因子抗原(colonization factor antigens,CFAs)和腸毒素(enterotoxins)參與了腹瀉過程。ETEC菌體表面的菌毛(又稱定居因子抗原)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腸上皮細(xì)胞,經(jīng)定居、繁殖產(chǎn)生腸毒素而致病。迄今為止,了解得比較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是一種優(yōu)勢血清型定居因子,其相應(yīng)抗體在阻止病原體在宿主小腸定居而引起腹瀉方面起重要怍用,在許多地方流行腹瀉區(qū),它是許多疫苗的一個重要組成成分。腸毒素是ETEC產(chǎn)生的毒性分泌蛋白,有不耐熱腸毒素(heat—labile entemtoxin,LT)和耐熱腸毒素(heat—stable entemtoxin,sT)兩種。CT和LT有很強(qiáng)的免疫原性和佐劑活性。LTB為無毒性的LT的B亞單位,也和CTB一樣有很強(qiáng)的免疫原性和佐劑活性。本研究中選用LTB作為粘膜免疫佐劑。 ETEC疫苗要求能夠中和大多數(shù)ETEC菌株的毒力因子抗原,目前普遍認(rèn)為一個合理的大腸桿菌疫苗應(yīng)該包括三個主要的菌毛抗原即CFA/I,CFA/II,和CFA/IV以及具有免疫原性的不耐熱腸毒素LT。目前ETEC研究熱點(diǎn)集中在三個方向:1.免疫方式由傳統(tǒng)的肌肉、皮下等轉(zhuǎn)為粘膜免疫;2.尋求粘膜佐劑,常用CT和LT;3.表達(dá)載體由傳統(tǒng)的有毒轉(zhuǎn)向減毒或無毒載體。 雙歧桿菌是人類腸道的自然宿主且可以粘附于腸道上皮細(xì)胞。因此,本研究的目的是將雙歧桿菌發(fā)展成一個表達(dá)CFA/I的口服活疫苗的抗原表達(dá)系統(tǒng)。 目的:構(gòu)建攜帶ETEC CFA/I的雙歧桿菌重組疫苗(即pBEX-CFA/I),然后將此載體疫苗免疫SD大鼠,檢測其在大鼠體內(nèi)誘導(dǎo)的體液和粘膜免疫應(yīng)答,并與雙歧桿菌重組LTB(即pBES-LTB)共免疫,檢測其粘膜免疫佐劑的效應(yīng)。 方法:(1)以pGEX-5x-1為基礎(chǔ),構(gòu)建穿梭表達(dá)載體pBEX-CFA/I。將其電轉(zhuǎn)化嬰兒雙歧桿菌,SDS-PAGE驗(yàn)證蛋白的表達(dá)。通過家兔腸袢實(shí)驗(yàn)驗(yàn)證表達(dá)蛋白的安全性。(2)用雙歧桿菌重組載體疫苗免疫SD大鼠:隨機(jī)分四組分別為PBS、pBES-LTB、pBEX-CFA/I和pBES-LTB+pBEX-CFA/I組,每組12只,免疫三次(0,10,17天),并于0,7,10,14,17,22和27天采血和糞便樣本,ELISA檢測其特異抗體水平。(3)在第27天,每組一半大鼠腹腔感染致死劑量ETEC毒株H10407,連續(xù)觀察20天,計算其存活力。另一半鼻飼ETECH10407,觀察其肺部感染情況。 結(jié)果:(1)CFA/I蛋白在雙歧桿菌中成功表達(dá),其表達(dá)蛋白經(jīng)家兔腸袢實(shí)驗(yàn)證實(shí)是無毒的。(2)ELISA結(jié)果表明:pBEX-CFA/I與pBES- LTB疫苗聯(lián)合免疫組的大鼠比其它單獨(dú)免疫組產(chǎn)生了更強(qiáng)烈的血清IgG和糞便IgA抗體(P0.05)。CFA/I免疫組和LTB免疫組間無顯著性差別(P0.05)。(3)腹腔攻毒保護(hù)實(shí)驗(yàn)結(jié)果證實(shí), pBEX-CFA/I + pBES-LTB免疫組的SD大鼠保護(hù)性比單獨(dú)免疫組的好,單獨(dú)口服天然雙歧桿菌和PBS的未免疫組無保護(hù)性。(4)ETEC鼻飼實(shí)驗(yàn)結(jié)果表明:pBEX-CFA/I+pBES-LTB免疫組及pBEX-CFA/I單獨(dú)免疫的SD大鼠肺部均無病理變化。 結(jié)論:(1)雙歧桿菌可以作為ETEC重組口服活疫苗的表達(dá)載體系統(tǒng),該口服疫苗表達(dá)系統(tǒng)開辟了ETEC疫苗研究的新方向;(2)重組雙歧桿菌表達(dá)-CFA/I單獨(dú)接種對ETEC的粘附和感染有免疫保護(hù)作用;(3)雙歧桿菌-CFA/I和雙歧桿菌-LTB聯(lián)合免疫,可明顯提高CFA/I的抗體滴度,并使動物獲得更好的保護(hù)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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