【作者】 罗耀玲；

【导师】 宋方洲； 马永平；

【作者基本信息】 重庆医科大学 ， 生物化学与分子生物学， 2008， 硕士

【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(P<0.05)。LTB免疫组和CFA/I免疫组间差别无统计学意义(P>0.05)。(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。更多还原

【Abstract】 Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease worldwide. It remains major causes of diarrheal morbidity and infant mortality in developing countries, and is also pathogenic bacteria for travelers diarrhea, and perennially associated with disease in soldiers deployed to developing countries. So a safe and effective vaccine against ETEC would be important to public health.ETEC are noninvasive and colonize the small intestines by attachment to mucosa via colonization factors (CFA). Then induce fluid and electrolyte secretion by the small intestinal epithelium in response to the production of heat-labile (LT) or heat-stable (ST) enterotoxins. CFAs are fimbrial adhesins that promote attachment to intestinal epithelium and known to provide protection against infection with ETEC expressing homologous CFs. The most common CFAs are CFA/I; In many areas of endemicity CFA/I is one of the most common CFA expressed by ETEC and so represents an important component of any vaccine. ST is encoded by the estA gene and is a monomeric polypeptide of 19 amino acids and has poorly immunogenic. ETEC strains that express CFA/I almost always express ST. LT is encoded by the eltAB,consisting of a toxic A subunit (LTA) and a pentamer of receptor-binding B subunits (LTB) similar to cholera toxin (CT). CT and LT are reported powerful immunogens and adjuvants and same to the CTB and LTB.ETEC vaccine requires the targeting of virulence factors or antigens common to large numbers of ETEC strains. There is general agreement that a logical vaccine strategy for ETEC would target the three major CFAs of ETEC (i.e.CFA/I, CFA/II, and CFA/IV) as well as the immunogenic LT. Today, the strategies for ETEC vaccine are focused on the three pathways: (a) to enhance traditional vaccine immunity by adding new adjuvants; (b) to develop mucosal immune vaccine and mucosal immune pathway; (c) to develop attenuated or atoxigenic vector expressing ETEC antigens. Bifidobacteria are natural inhabitants of the human intestinal tract and can adhere to the gut. We try to develop it as a gastrointestinal tract administers live vaccine system carrying CFA and LTB of ETEC.Objective: We aimed to construct a bifidobacteria based vaccine expression ETEC CFA/I and LTB, then to analyze its immunity by test the specific antibodies of CFA/I and LTB in SD rats post the vaccine immunize and by measure the survival rate post challenge with ETEC H10407.Methods: (1)Ltb gene was cloned to a shuttle expression vector pBES which originated from plasmid pBV220. This vector was named pBES-LTB and transformed into B. infantis. The product was tested by SDS-PAGE and its safety test by ansa intestinalis test .(2)Immunizing SD rat by recombined B. infantis–LTB (or CFA/I) vaccine. The rats were randomly placed in four groups of 12: group 1:PBS, group 2: pBES-LTB , group 3. pBES- CFA/I ,group 4:B. pBES-LTB + pBES- CFA/I. The above groups of rats were immunized intragastricly three times at 0d, 10d and 17d. Blood and fecal pellets were collected from rats at 0d, 7d, 10d, 14d, 17d, 22d and 27d. Specific antibodies were detected in sera and fecal pellets of rats by ELISA. (3) At 27d, half of SD rats in each group were challenged with a lethal dose of ETEC strain H10407 through abdominal cavity injection, and counting the mortality; Others were challenged intranasally with ETEC H10407(2×1010CFU).Results: (1) The ltb gene could stable expressed in B. infantis and the product (LTB) has a gentle toxicity of inducing intestinal juice secretory in rabbit by ansa intestinalis test. (2) The LTB、the CFA/I and the two antigens co-immunization all induced strong serum IgG and fecal IgA . The serum IgG and fecal IgA antibody from group 4 (oral inoculation with bifidobacteria–LTB + bifidobacteria–CFA/I) were significantly greater (P<0.05) than the antibody from other groups; But there were no significantly greater (P>0.05) between the group 2 (LTB) and the group 3 (CFA/I). (3) In the experiment of immunization protection assays , the SD rats got a significant protection, which immunized with bifidobacteria -LTB + bifidobacteria -CFA/I. But the LTB group and PBS group had no protection.Conclusion: (1)Bifidobacteria can be developed as oral vaccine expression system. (2) The bifidobacteria-LTB can induce specific antibodies via oral immune. (3) When bifidobacteria-LTB co- a bifidobacteria-LTB vaccine enhances bifidobacteria-CFA/I immune responses significantly as well as improves the survival rate of SD rats challenged by ETEC. This recombined bifidobacteria based vaccine pave a smooth way for ETEC vaccine development.更多还原