本文選題：產(chǎn)腸毒素大腸桿菌(ETEC) + 雙歧桿菌��； 參考：《重慶醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 在世界范圍內(nèi),產(chǎn)腸毒素大腸桿菌(ETEC)感染是引起腹瀉最重要的因素之一。它是發(fā)展中國(guó)家腹瀉發(fā)病和嬰兒死亡的主要原因,也是旅行者腹瀉與國(guó)家士兵腹瀉的最常見(jiàn)病原菌。因此,一個(gè)安全有效的抵抗ETEC腹瀉的疫苗對(duì)公眾健康是非常重要的。 ETEC是非侵襲性的,是依靠定居因子(CFA)粘附小腸粘膜上的,進(jìn)而分泌腸毒素LT和ST。CFAs是細(xì)菌細(xì)胞表面的鞭毛,可以促進(jìn)對(duì)小腸上皮細(xì)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基因編碼的19個(gè)氨基酸組成的單體多肽,免疫原性很差。LT是由eltAB編碼,結(jié)構(gòu)與霍亂毒素(CT)相似。它由兩個(gè)亞單位組成即有毒性的A單位(LTA)和五聚體的B單位(LTB)組成。CT和LT有很強(qiáng)的免疫原性和粘膜佐劑活性。LTB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作為免疫原和粘膜免疫佐劑。 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(gè)合理的大腸桿菌疫苗應(yīng)該包括三個(gè)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。因此在研究LTB免疫活性時(shí)選用CFA/I為參照。目前ETEC疫苗研究熱點(diǎn)集中在三個(gè)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.免疫途徑上尋求粘膜佐劑,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(wú)毒載體。根據(jù)以上三點(diǎn),我們采用無(wú)毒的雙歧桿菌為表達(dá)載體,通過(guò)粘膜免疫SD大鼠,檢LTB的免疫原性,并與重組的雙歧桿菌-CFA/I疫苗共免疫以檢測(cè)其粘膜佐劑活性。 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(gè)表達(dá)LTB蛋白的口服活疫苗的抗原表達(dá)系統(tǒng)。 目的:構(gòu)建攜帶ETEC LTB的雙歧桿菌重組疫苗,然后將此載體疫苗免疫SD大鼠,檢測(cè)其在大鼠體內(nèi)誘導(dǎo)的體液和粘膜免疫應(yīng)答,并與雙歧桿菌重組的pBES-CFA/I疫苗共免疫,檢測(cè)其粘膜免疫佐劑的效應(yīng)。 方法:(1)以pBV220為基礎(chǔ),構(gòu)建穿梭表達(dá)載體pBES-LTB。將其電轉(zhuǎn)化嬰兒雙歧桿菌,SDS-PAGE驗(yàn)證蛋白的表達(dá),并通過(guò)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、pBES-CFA/I和pBES-LTB+PBES-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(cè)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(jì)算其存活力。另一半鼻飼ETEC H10407,觀察其肺部感染情況。 結(jié)果:(1)LTB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)是微毒的。(2)ELISA結(jié)果表明:pBES-LTB與pBES-CFA/I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)。LTB免疫組和CFA/I免疫組間差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(3)腹腔攻毒保護(hù)實(shí)驗(yàn)結(jié)果證實(shí), pBES-LTB + pBES-CFA/I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的免疫組無(wú)保護(hù)性。(4)ETEC鼻飼實(shí)驗(yàn)結(jié)果表明:pBES-LTB +pBES-CFA/I免疫組及pBES-CFA/I單獨(dú)免疫的SD大鼠肺部均無(wú)病理變化, pBES-LTB免疫組有一定的炎癥反應(yīng),未免疫組肺部有嚴(yán)重的病理變化。 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(kāi)辟了ETEC疫苗研究的新方向;(2)雙歧桿菌表達(dá)的LTB單獨(dú)接種對(duì)ETEC的粘附無(wú)免疫保護(hù)性,但腸袢試驗(yàn)證明它對(duì)LT具有免疫性;(3)pBES-LTB與pBES-CFA/I聯(lián)合免疫,可明顯提高CFA/I的抗體滴度,并使動(dòng)物獲得更好的保護(hù)力,即具備口服疫苗免疫佐劑的基本特性。
[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.

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

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