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
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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。
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.
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。
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.
引文
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[13] Fingerut E, Gutter B, Goldway M, et a1. B subunit of E. coli enterotoxin as adjuvant and carrier in oral and skin vaccination[J]. Vet Immunol Immunopathol.2006,112:253~263
[14] Hou X, Liu JE. Construction of Escherichia coli-Bifidobacterium longum shuttle vector and expression of tumor suppressor gene PTEN in B. longum [ J].Wei Sheng Wu Xue Bao, 2006 ,46(3):347~352
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[18] 房海 主编.大肠埃希氏菌[M].河北科学技术出版社,1997,133~143
[19] Byrd W,Cassels FJ. Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heatlabile enterotoxin[J]. Vaccine.2003,21:1884~1893
[20] 朱立平,陈学清 主编 . 免疫学常用实验方法 [M]. 人民军医出版社 , 1999.352~355
[21] 施新猷 主编.现代医学实验动物学[M].人民军医出版社.1999. 298~342
[22] 王志斌,姜普林,曾年华等.肠毒素大肠杆菌攻毒小鼠模型的建立以及疫苗候选株保护效果的评价[J].世界华人消化杂志.2006,14(28):2753~2758
[23] Ang D,Christopher M E,Nagata L P,et a1.Intranasal immunization with liposom-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicits mucosal-cellular and humoral immune responses[J].J Clin V iro 1.2004,31(Suppl 1):S99~S106
[24] Vajdy M,Hagan D T. Microparticles for intranasal immunization[J].Adv Drug Delw Rev.2001,51(1~3):127
[25] 陈志华,邢丽,石辛甫,等.双价痢疾工程疫苗经粘膜免疫动物的实验研究[J].中华微生物学和免疫学杂志.2001,5:87~92
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[27] Robin McKenzie,A. Louis Bourgeois, Fayette Engstrom, et al. Comparative Safety and Immunogenicity of Two Attenuated Enterotoxigenic Escherichia coli Vaccine Strains in Healthy Adults[J]. infection and immunity. 2006, 74(2):994~1000
[28] Pascual DW, Hone DM, Hall S, et al. Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response[J]. Infect Immun. 1999 ,67(12):6249~6256
[29] Tacket CO,Pasetti MF,Edelman R,et at.Immunogenicity of recombinant LT-B delivered orally to humans in transgenic com[J].Vaccine.2004,22(31/32):4 385~89
[30] Rosales-Mendoza S, Soria-Guerra RE, López-Revilla R, et at.Ingestion of transgenic carrots expressing the Escherichia coli heat-labile enterotoxin B subunit protects mice against cholera toxin challenge[J].Plant Cell Rep. 2008 ,27(1):79~84
[31] Hearn A R,Haan L,Pemberton A J,et a1.Tramcking of exogenous peptides into proteasome-dependent major histocompatibility complex class I pathway following enterotoxln B subunit—mediated delivery[J].J Biol Chem.2004,279:51315~51322
[32] Apostolaki M,Williams N A.Nassl delivery of antigen with the B subunit of Escherichia coli heat-labile enterotoxin augments antigen-specific T-cell clonal expansion and differention[J].Infect Immun.2004,72:(l)4072~4080
[33] Turcanu V,Hirst T R,Williams N A.Modulation of human Monocytes by Escherichia coli heat-labile enterotoxin B-subunit:altered cytokine production and its functional consequences[J].Immunology,2002,106(3)l:316~325
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[1] Brandtzaeg P,Berstad A E,Farstad I N.Mueosal immunity-amajor adaptive defence mechanism[J].Behriug Inst Mitt.1997,98(1):81~84
[2] Ang D,Christopher M E,Nagata L P,et a1.Intranasal immunization with liposom-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicits mucosal-cellular and humoral immune responses[J].J Clin V iro 1.2004,31(Suppl 1):S99~S106
[3] 郝生宏,杨荣芳.粘膜免疫[J].营养与疾病.2004,4:42~44
[4] Brayden DJ,Jepson MA,Baird AW .Keynote review:intestinal Peyer’s patch M cells and oral vaccine targeting[J].Drug Diseov Today.2005,10:l145~1157
[5] Mowat,A M.Anatomical basis of tolerance and immunity to intestinal antigens[J].Nat Rev Immuno1.2003,3:33l~34l
[6] Hamada H,Hiroi T,Nishiyama Y.et a1.Identification 0f multiple isolated lymphoidfollicles on the antimesenteric wall of the inouse small intestine[J].J Immunol.2002,168:57~64
[7] Novak N, Haberstok J, Bieber T, Allam JP. The immune privilege of the oral mucosa[J]. Trends Mol Med. 2008 Apr 4 [in print]
[8] Goni F, Prelli F, Schreiber F, High titers of mucosal and systemic anti-PrP antibodies abrogate oral prion infection in mucosal-vaccinated mice[J]. Neuroscience. 2008 Mar 6 [in print]
[9] 陈志华,邢丽,石辛甫,等.双价痢疾工程疫苗经粘膜免疫动物的实验研究[J].中华微生物学和免疫学杂志.2001,5:87~92
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[11] Bo rsutzky S,Moreli V,Ebensen T,et a1.Eficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MAII-2 as adjuvant[J].Eur J Immunol.2003,33(6):1548~1556
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[13] Campos IB, Darrieux M, Ferreira DM, et a1.Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge[J]. Microbes Infect. 2008 Jan 20 [in print]
[14] Tafaghodi M, Jaafari MR, Tabassi SA. Nasal Immunization Studies by Cationic, Fusogenic and Cationic-Fusogenic Lipo-somes Encapsulated with Tetanus Toxoid[J]. Curr Drug Deliv. 2008,5(2):108~113
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[7] Pascual DW, Hone DM, Hall S, et al. Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response[J]. Infect Immun. 1999 ,67(12):6249~6256
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[11] 易 成,郭志英,黄英,et a1. 婴儿双歧杆菌介导的CD/5一FC自杀基因系统对黑色素瘤的抑瘤实验[J]. 四川大学医学版.2005,36(2):l65~l68
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[14] Hou X, Liu JE. Construction of Escherichia coli-Bifidobacterium longum shuttle vector and expression of tumor suppressor gene PTEN in B. longum [ J].Wei Sheng Wu Xue Bao, 2006 ,46(3):347~352
[15] Li X, Fu GF, Fan YR, et a1.Bifidobacterium adolescentis as a delivery system of endostatin for cancer gene therapy: selective inhibitor of angiogenesis and hypoxic tumor growth[J].Cancer Gene Ther. 2003,10(2):105~111
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[18] 房海 主编.大肠埃希氏菌[M].河北科学技术出版社,1997,133~143
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[20] 朱立平,陈学清 主编 . 免疫学常用实验方法 [M]. 人民军医出版社 , 1999.352~355
[21] 施新猷 主编.现代医学实验动物学[M].人民军医出版社.1999. 298~342
[22] 王志斌,姜普林,曾年华等.肠毒素大肠杆菌攻毒小鼠模型的建立以及疫苗候选株保护效果的评价[J].世界华人消化杂志.2006,14(28):2753~2758
[23] Ang D,Christopher M E,Nagata L P,et a1.Intranasal immunization with liposom-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicits mucosal-cellular and humoral immune responses[J].J Clin V iro 1.2004,31(Suppl 1):S99~S106
[24] Vajdy M,Hagan D T. Microparticles for intranasal immunization[J].Adv Drug Delw Rev.2001,51(1~3):127
[25] 陈志华,邢丽,石辛甫,等.双价痢疾工程疫苗经粘膜免疫动物的实验研究[J].中华微生物学和免疫学杂志.2001,5:87~92
[26] Byrd W,Cassels FJ. Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heat-labile enterotoxin[J]. Vaccine. 2003,21:1884~1893
[27] Robin McKenzie,A. Louis Bourgeois, Fayette Engstrom, et al. Comparative Safety and Immunogenicity of Two Attenuated Enterotoxigenic Escherichia coli Vaccine Strains in Healthy Adults[J]. infection and immunity. 2006, 74(2):994~1000
[28] Pascual DW, Hone DM, Hall S, et al. Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response[J]. Infect Immun. 1999 ,67(12):6249~6256
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[30] Rosales-Mendoza S, Soria-Guerra RE, López-Revilla R, et at.Ingestion of transgenic carrots expressing the Escherichia coli heat-labile enterotoxin B subunit protects mice against cholera toxin challenge[J].Plant Cell Rep. 2008 ,27(1):79~84
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[33] Turcanu V,Hirst T R,Williams N A.Modulation of human Monocytes by Escherichia coli heat-labile enterotoxin B-subunit:altered cytokine production and its functional consequences[J].Immunology,2002,106(3)l:316~325
[34] Alves AM, Lásaro MO, Almeida DF, et a1.New vaccine strategies against enterotoxigenic Escherichia coli. I: DNA vaccines against the CFA/I fimbrialadhesin.Braz J Med Biol Res. 1999,32(2):223~229
[35] Pascual DW, Hone DM, Hall S, et a1.Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response.Infect Immun. 1999,67(12):6249~6256
[36] Kenneth P. Allen,1Mildred M. Randolph, and James M. Fleckenstein. Importance of Heat-Labile Enterotoxin in Colonization of the Adult Mouse Small Intestine by Human Enterotoxigenic Escherichia coli Strains[J]. infection and immunity, 2006, 74(2):869~875
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[42] Binsztein N., M.J.Jouve, G.I.Viboud, et al. Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina[J]. J. Clin. Microbiol.1991,29:1893~1898
[1] Brandtzaeg P,Berstad A E,Farstad I N.Mueosal immunity-amajor adaptive defence mechanism[J].Behriug Inst Mitt.1997,98(1):81~84
[2] Ang D,Christopher M E,Nagata L P,et a1.Intranasal immunization with liposom-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicits mucosal-cellular and humoral immune responses[J].J Clin V iro 1.2004,31(Suppl 1):S99~S106
[3] 郝生宏,杨荣芳.粘膜免疫[J].营养与疾病.2004,4:42~44
[4] Brayden DJ,Jepson MA,Baird AW .Keynote review:intestinal Peyer’s patch M cells and oral vaccine targeting[J].Drug Diseov Today.2005,10:l145~1157
[5] Mowat,A M.Anatomical basis of tolerance and immunity to intestinal antigens[J].Nat Rev Immuno1.2003,3:33l~34l
[6] Hamada H,Hiroi T,Nishiyama Y.et a1.Identification 0f multiple isolated lymphoidfollicles on the antimesenteric wall of the inouse small intestine[J].J Immunol.2002,168:57~64
[7] Novak N, Haberstok J, Bieber T, Allam JP. The immune privilege of the oral mucosa[J]. Trends Mol Med. 2008 Apr 4 [in print]
[8] Goni F, Prelli F, Schreiber F, High titers of mucosal and systemic anti-PrP antibodies abrogate oral prion infection in mucosal-vaccinated mice[J]. Neuroscience. 2008 Mar 6 [in print]
[9] 陈志华,邢丽,石辛甫,等.双价痢疾工程疫苗经粘膜免疫动物的实验研究[J].中华微生物学和免疫学杂志.2001,5:87~92
[10] Pearay L Ogra.Mucosal immunity:Some historcal perpective on host-pathogen interactions and implications for mucosal vaccines[J].Immunology and cell biology.2003,81:23~33
[11] Bo rsutzky S,Moreli V,Ebensen T,et a1.Eficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MAII-2 as adjuvant[J].Eur J Immunol.2003,33(6):1548~1556
[12] 崔 萍,于三科.粘膜免疫佐剂及免疫途径研究进展[J].动物医学进展.2007,28(2):81~88
[13] Campos IB, Darrieux M, Ferreira DM, et a1.Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge[J]. Microbes Infect. 2008 Jan 20 [in print]
[14] Tafaghodi M, Jaafari MR, Tabassi SA. Nasal Immunization Studies by Cationic, Fusogenic and Cationic-Fusogenic Lipo-somes Encapsulated with Tetanus Toxoid[J]. Curr Drug Deliv. 2008,5(2):108~113
[15] 吴超,冯强,曾韦锟等. 重组大肠杆菌不耐热肠毒素B亚单位的表达、纯化及粘膜佐剂活性研究[J].中华微生物学和免疫学杂志.2005,25(4):307~311
[16] Byrd W,Cassels FJ. Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heat-labile enterotoxin[J]. Vaccine. 2003,21:1884~1893
[17] Van der Lubben I M,Verhoef J C,Bombard G,et a1.Chitosan for mucosal vaccinatlon[J].Adv Drug Deliv Rev.2001,52(2):139~143
[18] Vajdy M,Hagan D T.Microparticles for intranasal immunization[J].Adv Drug Delw Rev. 2001,51(1~3):127