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
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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(P<0.05)。CFA/I免疫组和LTB免疫组间无显著性差别(P>0.05)。(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。
Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease worldwide. It remains the first major causes of diarrheal morbidity and infant mortality in developing countries (second to Rotavirus), and is also pathogenic bacteria for travelers diarrhea, and perennially. So a safe and effective vaccine against ETEC would be important to public health.
It is already known that colonization factor antigens (CFAs) and enterotoxins of ETEC led to diarrheal. CFAs are fimbrial adhesins that promote attachment to intestinal epithelium and known to provide protection against infection with ETEC expressing homologous CFAs, ETEC are noninvasive and colonize the small intestines by attachment to mucosa via colonization factors (CFAs). The most common CFAs are the CFA/I, CFA/II and CFA/IV family. In many areas of endemicity, CFA/I is one of the most common factor expressed by ETEC and so represents an important component of any vaccine. Enterotoxins induce fluid and electrolyte secretion by the small intestinal epithelium in response to the production of heat-labile (LT) or heat-stable (ST) enterotoxins. 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 administer live vaccine system carrying CFA and LTB of ETEC.
Objective: We aimed to construct a bifidobacterium 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)CFA/I gene was cloned to a shuttle expression vector pBEX which originated from plasmid pGEX-5x-1. This vector was named pBEX-CFA/I 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 bifidobacterium–CFA/I vaccine.the rats were randomly placed in four groups of 12: group 1:PBS, group 2: bifidobacterium–CFA/I, group 3:bifidobacterium–LTB ,group 4: bifidobacterium–LTB + bifidobacterium-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 CFA/I gene could stable expressed in B. infantis and the product CFA/I has no toxicity of inducing intestinal juice secretory in rabbit by ansa intestinalis test. (2) The CFA/I, the LTB 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 bifidobacterium–CFA/I + bifidobacterium–LTB) 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 bifidobacterium-LTB + bifidobacterium-CFA/I. But the LTB group and PBS group had no protection.
Conclusion: (1)Bifidobacterium can be developed as oral vaccine expression system. (2) The bifidobacterium- CFA/I can induce specific antibodies via oral immune. (3) When co-administered with the bifidobacterium–LTB, the bifidobacterium- LTB vaccine enhances bifidobacterium–CFA/I immune responses significantly as well as improves the survival rate of SD rats challenged by ETEC. This recombined bifidobacterial based vaccine pave a smooth way for ETEC vaccine development.
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(P<0.05)。CFA/I免疫组和LTB免疫组间无显著性差别(P>0.05)。(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。
Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease worldwide. It remains the first major causes of diarrheal morbidity and infant mortality in developing countries (second to Rotavirus), and is also pathogenic bacteria for travelers diarrhea, and perennially. So a safe and effective vaccine against ETEC would be important to public health.
It is already known that colonization factor antigens (CFAs) and enterotoxins of ETEC led to diarrheal. CFAs are fimbrial adhesins that promote attachment to intestinal epithelium and known to provide protection against infection with ETEC expressing homologous CFAs, ETEC are noninvasive and colonize the small intestines by attachment to mucosa via colonization factors (CFAs). The most common CFAs are the CFA/I, CFA/II and CFA/IV family. In many areas of endemicity, CFA/I is one of the most common factor expressed by ETEC and so represents an important component of any vaccine. Enterotoxins induce fluid and electrolyte secretion by the small intestinal epithelium in response to the production of heat-labile (LT) or heat-stable (ST) enterotoxins. 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 administer live vaccine system carrying CFA and LTB of ETEC.
Objective: We aimed to construct a bifidobacterium 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)CFA/I gene was cloned to a shuttle expression vector pBEX which originated from plasmid pGEX-5x-1. This vector was named pBEX-CFA/I 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 bifidobacterium–CFA/I vaccine.the rats were randomly placed in four groups of 12: group 1:PBS, group 2: bifidobacterium–CFA/I, group 3:bifidobacterium–LTB ,group 4: bifidobacterium–LTB + bifidobacterium-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 CFA/I gene could stable expressed in B. infantis and the product CFA/I has no toxicity of inducing intestinal juice secretory in rabbit by ansa intestinalis test. (2) The CFA/I, the LTB 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 bifidobacterium–CFA/I + bifidobacterium–LTB) 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 bifidobacterium-LTB + bifidobacterium-CFA/I. But the LTB group and PBS group had no protection.
Conclusion: (1)Bifidobacterium can be developed as oral vaccine expression system. (2) The bifidobacterium- CFA/I can induce specific antibodies via oral immune. (3) When co-administered with the bifidobacterium–LTB, the bifidobacterium- LTB vaccine enhances bifidobacterium–CFA/I immune responses significantly as well as improves the survival rate of SD rats challenged by ETEC. This recombined bifidobacterial based vaccine pave a smooth way for ETEC vaccine development.
引文
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[28] 施新猷 主编.现代医学实验动物学[M].人民军医出版社.1999,p298-342
[29] 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
[30] 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
[31] Eric R. Hall,Thomas F. Wierzba,Christina ?hrén,et al. Induction of Systemic Antifimbria and Antitoxin Antibody Responses in Egyptian Children and Adults by an Oral, Killed Enterotoxigenic Escherichia coli plus Cholera Toxin B Subunit Vaccine [J].Infect Immun. 2001, 69(5): 2853–2857
[32] Barry,E.M.,Z.Altboum, G.A.Losonsky, and M. M. Levine. Immune responses elicited against multiple fimbriae and mutant LT from enterotoxigenic E. coliexpressed in attenuated Shigella vaccine strains[J].Vaccine.2003,21:333-340
[33] Piller KJ,Clemente TE,Jun SM,et al. Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean[J] .Planta.2005,222(1):6-18
[34] Rosales-Mendoza S,Soria-Guerra RE,López-Revilla R,et al.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
[35] Vajdy M,Hagan D T.Microparticles for intranasal immunization[J].Adv Drug Delw Rev.2001,51(1-3):127
[36] 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
[37] 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
[38] Arthur K. Turner,Juliet C. Beavis,Jonathan C. Stephens, et a1.Construction and Phase I Clinical Evaluation of the Safety and Immunogenicity of a Candidate Enterotoxigenic Escherichia coli Vaccine Strain Expressing Colonization Factor Antigen CFA/I[J]. Infection and Immunity.2006,74(2):1062–1071
[39] Wyatt Byrd & Frederick J. Cassels. Long-term systemic and mucosal antibody responses measured in BALB/c mice following intranasal challenge with viable Enterotoxigenic Escherichia coli[J].FEMS Immunol Med Microbiol.2006,46:262–268
[40] Cravioto A,Reyes RE,Trujillo F,et al.Risk of diarrhea during the first year of life associated with initial and subsequent colonization by soecific enteropathogens[J].Am J Epidemiol.1990,131:886-904
[41] Levine MM,Nalin DR,Hoover DL,et al.Immunity to Enterotoxigenic Escherichia coli[J].Infection and immunity.1979,23:729-736
[42] Binsztein N, M.J.Jouve, G.I.Viboud, et al. Colonization factors of enterotoxigenicEscherichia coli isolated from children with diarrhea in Argentina[J].Clin. Microbiol. 1991,29:1893-1898
[1] 田洪涛,张篪,王占武,等.影响双歧杆菌微生态制剂菌体存活的因素[J].中国乳品工业.2000,28(1):23—25
[2] 康白.正常微生物群是一个新的人体生理学系统[J].中国微生态学杂志.2003,15(2):63—65
[3] 张风云,王忠海.双歧杆菌的生理作用及临床应用的研究进展[J].临床荟萃.2004,19(15):892—893
[4] 粱冰,吴力克,等.双歧杆菌QJ405、乳杆菌QJ405在试管内对肠道致病菌抑杀效果的初步报告[J].中国微生态学杂志.2000,12(5):269—271
[5] 张宝元,柳军,马晓红.双歧杆菌在儿科临床及保健中的应用[J].北京医学.2001,23:63—64
[6] Tejada—SimonMV,UstunolZ,PestkaJJ.Ex vivo efects of 1actobacilli,streptococci,and bifidobacteria ingestion on cytokine and nitric oxide production in a murinemode1.J Food Prot.1999,62:162—169.
[7] 穆小萍,张德纯.双歧杆菌的免疫调节作用研究现状与展望[J].中国微生态学杂志.2007(1):115—117
[8] 汪雪雁.双歧杆菌的研究进展[J].安徽农业大学学报.2000,27(2):175—177
[9] 陈文学,史俊华.双歧杆菌的研究进展[J].中国微生态学杂志.2000,12:298—300
[10] Yamazaki S,et a1.2-Amino-3-carboxy 1,4-naphthoquinone afects the end product profile of bifido-bacteria through the mediated oxidation of NAD(P)H[J].Appl Microbiol Biotechnol.2002,59(1):72.
[11]李迎雪,王立生.双歧杆菌作为基因治疗转移载体的研究进展[J].中国微生态学杂志.2006,18(1):76-77
[12] 吴瑜,易成,王树人,等.婴儿双歧杆菌对小鼠黑色素瘤模型肿瘤组织的靶向性[J].四川大学学报(医学版).2003,34(3):435— 43
[13] 郭志英,易成,王树人,等.婴儿双歧杆菌胞嘧啶脱氨酶肿瘤靶向性基因治疗系统的构建[J].中国肺癌杂志.2004,7(2):95— 98
[14] 徐根兴.转人实体瘤内皮抑制因子基因双歧杆菌的方法[J].发明专利公报.1999,15(20):20
[15]易力,倪学勤,藩康成,等. 双歧杆菌的研究进展[J]. 兽药与饲料添加剂.2005,10(1):22
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[4] Mailing Jertborn,Christina Ahren,Ann-mari,SVENNERHOLM1Dose-Dependent Circulating Immunoglobulin A Antibody-Secreting Cell and Serum Antibody Responses in Swedish Volunteers to an Oral Inactivated Enterotoxigenic Escherichia coli Vaccine[J].Clinical and Diagnostic Laboratory Immunology. 2001,8(2): 424~428
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[22] 吕晓英, 张朝武. 乳酸杆菌及双歧菌基因表达载体系统的研究近况[J]. 现代预防医学.2005,32(3):213-125
[23] 李迎雪,王立生. 双歧杆菌作为基因治疗转移载体的研究进展[J]. Chinese Journal of Microecolog.2006,18(1):76-77
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[27] 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,p352-355
[28] 施新猷 主编.现代医学实验动物学[M].人民军医出版社.1999,p298-342
[29] 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
[30] 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
[31] Eric R. Hall,Thomas F. Wierzba,Christina ?hrén,et al. Induction of Systemic Antifimbria and Antitoxin Antibody Responses in Egyptian Children and Adults by an Oral, Killed Enterotoxigenic Escherichia coli plus Cholera Toxin B Subunit Vaccine [J].Infect Immun. 2001, 69(5): 2853–2857
[32] Barry,E.M.,Z.Altboum, G.A.Losonsky, and M. M. Levine. Immune responses elicited against multiple fimbriae and mutant LT from enterotoxigenic E. coliexpressed in attenuated Shigella vaccine strains[J].Vaccine.2003,21:333-340
[33] Piller KJ,Clemente TE,Jun SM,et al. Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean[J] .Planta.2005,222(1):6-18
[34] Rosales-Mendoza S,Soria-Guerra RE,López-Revilla R,et al.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
[35] Vajdy M,Hagan D T.Microparticles for intranasal immunization[J].Adv Drug Delw Rev.2001,51(1-3):127
[36] 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
[37] 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
[38] Arthur K. Turner,Juliet C. Beavis,Jonathan C. Stephens, et a1.Construction and Phase I Clinical Evaluation of the Safety and Immunogenicity of a Candidate Enterotoxigenic Escherichia coli Vaccine Strain Expressing Colonization Factor Antigen CFA/I[J]. Infection and Immunity.2006,74(2):1062–1071
[39] Wyatt Byrd & Frederick J. Cassels. Long-term systemic and mucosal antibody responses measured in BALB/c mice following intranasal challenge with viable Enterotoxigenic Escherichia coli[J].FEMS Immunol Med Microbiol.2006,46:262–268
[40] Cravioto A,Reyes RE,Trujillo F,et al.Risk of diarrhea during the first year of life associated with initial and subsequent colonization by soecific enteropathogens[J].Am J Epidemiol.1990,131:886-904
[41] Levine MM,Nalin DR,Hoover DL,et al.Immunity to Enterotoxigenic Escherichia coli[J].Infection and immunity.1979,23:729-736
[42] Binsztein N, M.J.Jouve, G.I.Viboud, et al. Colonization factors of enterotoxigenicEscherichia coli isolated from children with diarrhea in Argentina[J].Clin. Microbiol. 1991,29:1893-1898
[1] 田洪涛,张篪,王占武,等.影响双歧杆菌微生态制剂菌体存活的因素[J].中国乳品工业.2000,28(1):23—25
[2] 康白.正常微生物群是一个新的人体生理学系统[J].中国微生态学杂志.2003,15(2):63—65
[3] 张风云,王忠海.双歧杆菌的生理作用及临床应用的研究进展[J].临床荟萃.2004,19(15):892—893
[4] 粱冰,吴力克,等.双歧杆菌QJ405、乳杆菌QJ405在试管内对肠道致病菌抑杀效果的初步报告[J].中国微生态学杂志.2000,12(5):269—271
[5] 张宝元,柳军,马晓红.双歧杆菌在儿科临床及保健中的应用[J].北京医学.2001,23:63—64
[6] Tejada—SimonMV,UstunolZ,PestkaJJ.Ex vivo efects of 1actobacilli,streptococci,and bifidobacteria ingestion on cytokine and nitric oxide production in a murinemode1.J Food Prot.1999,62:162—169.
[7] 穆小萍,张德纯.双歧杆菌的免疫调节作用研究现状与展望[J].中国微生态学杂志.2007(1):115—117
[8] 汪雪雁.双歧杆菌的研究进展[J].安徽农业大学学报.2000,27(2):175—177
[9] 陈文学,史俊华.双歧杆菌的研究进展[J].中国微生态学杂志.2000,12:298—300
[10] Yamazaki S,et a1.2-Amino-3-carboxy 1,4-naphthoquinone afects the end product profile of bifido-bacteria through the mediated oxidation of NAD(P)H[J].Appl Microbiol Biotechnol.2002,59(1):72.
[11]李迎雪,王立生.双歧杆菌作为基因治疗转移载体的研究进展[J].中国微生态学杂志.2006,18(1):76-77
[12] 吴瑜,易成,王树人,等.婴儿双歧杆菌对小鼠黑色素瘤模型肿瘤组织的靶向性[J].四川大学学报(医学版).2003,34(3):435— 43
[13] 郭志英,易成,王树人,等.婴儿双歧杆菌胞嘧啶脱氨酶肿瘤靶向性基因治疗系统的构建[J].中国肺癌杂志.2004,7(2):95— 98
[14] 徐根兴.转人实体瘤内皮抑制因子基因双歧杆菌的方法[J].发明专利公报.1999,15(20):20
[15]易力,倪学勤,藩康成,等. 双歧杆菌的研究进展[J]. 兽药与饲料添加剂.2005,10(1):22
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