EHEC O157：H7 Stx2B-Tir-Stx1B-Zot重组菌的构建及其免疫原性的研究
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摘要
食源性病原微生物是当今世界上不断增多的食品安全和突发性公共卫生事件的主要诱因。其中,肠出血性大肠杆菌(enterohaemorrhagic Escherichia coli, EHEC)0157：H7可致人腹泻、出血性结肠炎、还可在5%-10%的病例中引发溶血性尿毒综合征(Hemolytic uremic syndrome, HUS)及血栓性血小板减少紫癜等严重并发症,严重者可致死亡。近20余年来,EHEC O157:H7引发的食物中毒在世界各地包括中国都有不同规模的暴发流行。
在我们研究中选用EHEC O157:H7三个主要的毒力基因tir. stx2b和stx1b,运用原核表达系统在体外进行串联表达,获得重组蛋白。选择能够增强肠通透的霍乱弧菌封闭小带毒素zot基因进行串联,希望能够增强机体的粘膜免疫,尤其是肠道的粘膜免疫。选用链霉素处理的Balb/c小鼠作为评价动物,皮下和鼻腔两个途径免疫Stx2B-Tir-Stx1B-Zot蛋白,通过测定血清中的IgG滴度和IgA滴度、粪便中的IgA滴度以及粪便排菌来衡量Stx2B-Tir-Stx1B-Zot蛋白的免疫原性,为EHEC O157:H7亚单位疫苗的研制筛选良好的免疫原。
1.肠出血性大肠杆菌0157：H7 tir基因重组蛋白的表达及鉴定
克隆、表达肠出血性大肠杆菌(enterohaemorrhagic Escherichia coli, EHEC)0157：H7转位紧密素受体(tir)基因,并研究其免疫原性。选用pET28原核表达载体,构建pET28-tir重组质粒,并在大肠杆菌BL21中进行高效表达。选用家兔制备高滴度多克隆抗体,Western blot分析其免疫原性。结果显示成功获得高效表达的重组Tir蛋白,并制备了兔源Tir多克隆抗体,Western blot分析此抗体能与EHEC O157:H7(?)勺天然Tir蛋白发生特异性抗原抗体反应。我们在大肠杆菌中成功克隆表达了tir基因,所获重组Tir蛋白具有良好的反应原性。
2.大肠杆菌O157:H7 Tir重组蛋白的生物学特性
以HEp-2细胞为模型,评价重组Tir蛋白制备的抗体是否能够抑制(?)EHEC O157:H7的粘附和A/E损伤的形成。应用纯化后的重组Tir蛋白,皮下免疫Balb/c小鼠进行免疫保护性实验。两免断尾采血,间接ELISA测定血清特异性抗体效价。二免14d,10’。CFUEHEC O157:H7口服灌胃攻毒。每隔两天采集粪便检测排菌量。结果显示：表达的Tir蛋白具有天然Tir蛋白的功能特性,其抗体能够较大程度地抑制EHEC 0157：H7对HEp-2细胞的粘附和A/E损伤的形成。二免Balb/c小鼠存活率高达100%,排菌减少,8d后检测不到排菌,而对照组在第16d仍然能检测到排菌。因此,体外表达的重组Tir蛋白具有良好的抗原性,可作为EHEC O157:H7基因工程疫苗研制的首选抗原之一
3.肠出血性大肠杆菌O157:H7 Stx2B-Tir-StxlB-Zot多价融合蛋白表达及鉴定
EHEC O157:H7主要定植在肠道内,研制能够增强粘膜免疫基因工程疫苗具有很大优势。构建表达tir和stxb的融合基因,将tir基因中间295个氨基酸残基(Tir295)与stXB亚基基因72个氨基酸串联构建ppGEX-stx2b-tir-stx1b重组质粒,并与霍乱弧菌封闭小带毒素(Zot)靠近C端基因串联,构建重组质粒pGEX-stx2b-tir-stxl b-zot,转化于BL21(DE3),用IPTG进行诱导表达,经SDS-PAGE电泳检测,该融合蛋白获得了高效表达,主要以可溶性形式存在于菌体胞浆中。Western blot鉴定显示,Stx2B-Tir-Stx1B-Zot具有良好的反应原性。
4.大肠杆菌O157:H7 Stx2B-Tir-StxlB-Zot重组蛋白的免疫原性
Stx2B-Tir-Stx1B-Zot重组蛋白,分别皮下和鼻腔免疫Balb/c小鼠,二次免疫后测定血清中IgG和IgA滴度,粪便中IgA滴度。定期采集粪便,测定粪便排菌情况。结果显示：鼻腔免疫后Balb/c小鼠存活率100%,攻毒7d后检测不到排菌,并且最高排菌量只有3×103CFU/0.1g,血清中IgG滴度接近105,粪便中IgA接近102；皮下免疫后Balb/c小鼠存活率88%,攻毒9d后检测不到排菌,并且最高排菌量只有1.1×105CFU/0.1g,血清中IgG滴度也接近105,粪便中检测不到IgAStx2B-Tir-Stx1B-Zot鼻腔免疫能够发挥更好的免疫原性,可以作为EHEC 0157：H7亚单位疫苗研制的免疫原。
Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic enteric pathogen of worldwide importance, and is one of Shiga toxin-producing Escherichia coli (STEC). The type III secretion system of E. coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E.coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXcp of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery.
In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-StxlB-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST'Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Our aim is to determine whether antibodies specific for Stx2B-Tir-Stx1B-Zot could prevent O157:H7 colonization, to determine the role of Zot in the blocking colonization as well as to develop the subunit vaccine for EHEC O157:H7.
1. Construction and Expression of Recombinant Protein Tir from Escherichia coli O157:H7
The objective of the experiment is to clone, express and study the antigenicity of translocation intimin receptor(tir) gene from EHEC O157:H7. The vector pET28 and BL21(DE3) were used to construct and overexpress the recombinant protein of tir gene by prokaryotic expression. The recombinant Tir protein was used to immunize rabbits to obtain high-titer polyclonal antibodies,which was used to analyze the antigenicity of Tir by Western blot. We obtain successfully high-level expression of recombinant Tir protein and the molecular weight is 48kD.The polyclonal antibody against Tir protein have good a reactive fragment with the native Tir from Escherichia coli O157:H7. In conclusion, tir gene was cloned and expressed successfully in Escherichia coli, and recombinant Tir protein has a good antigenicity.
2. Biological Characterization of Recombinant Tir Protein from Escherichia coli O157:H7
HEp-2 cell and the polyclonal anti-Tir were used to evaluate adherence, adherence inhibition and attaching and effacing lesion (A/E) of EHEC O157:H7. Balb/c mice were inoculated with purified 100μg Tir protein subcutaneously and the protection rate and fecal shedding were analyzed after orally challenged by 1010CFU EHEC O157:H7. Anti-Tir IgG titers were detected by indirect ELISA. Balb/c mice immunized twice showed survival rate up to 100% compared to the 50% survival rate. Moreover, no EHEC O157:H7 in the fecal samples was detected after 8d challenged with O157:H7,but non-immunized mice still sheded the organism on 16th day.The above-mentioned results indicated that Tir protein has a good antigenicity and could be used for EHEC 0157 genetic engineering vaccines.
3. Expression Stx2B-Tir-StxlB-Zot Multivalent fusion protein from EHEC O157:H7
EHEC O157:H7 mainly colonize the intestine tissue, therefore, developing the genetic engineering vaccine enhancing the mucosal immunity has a significant dominance. In this study, we amplified tir, stx1b and stx2b genes and contructed the recombinant plasmid of pGEX-stx2b-tir-stx1b. The positive pGEX-stx2b-tir-stx1b were digested with Xho I, dephosphorylated with alkaline phosphatase and ligated with the zot gene to construct the pGEX-stx2b-tir-stx1b-zot. BL21(pGEX-stx2b-tir-stx1b-zot) was induced with IPTG and analysize with SDS-PAGE. All the results indicated that the recombinant Stx2B-Tir-Stx1B-Zot was successfully expressed, the molecular weight is 107kD and the anti-Stx2B-Tir-Stx1B-Zot antibody are able to react with the native Stx2B,Tir and Stx1B from EHEC 0157:H7.
4. Immunogenicity Analysis of Stx2B-Tir-StxlB-Zot from EHEC O157:H7
Balb/c mice were immunized subcutaneously and intranasally with Stx2B-Tir-StxlB-Zot protein, respectively at week 0,3. After the second immunization, the blood from the immunized and naive mice were collected to prepare sera for detecting the IgG and IgA titers. The feces form the immunized and naive mice were collected to detect the secretory IgA titers. Balb/c mice immunized intranasally have 100% survival rate, no detectable bacteia on the 7th after challenged, the highest shedding of 3×103CFU/0.1g and 105 IgG titers in sera and aroud102IgA titers in feces. Balb/c mice immunized subcutanously have 88% survival rate, no detectable bacteia on the 9th after challenged, the highest shedding of 1.1×105CFU/0.1g and 105 IgG titers in sera and no IgA titers in feces. Stx2B-Tir-Stx1B-Zot immunized intranasally is capable to play better immogenicity and able to be a preferred antigen to develop the subunit vaccine for EHEC O157:H7.
在我们研究中选用EHEC O157:H7三个主要的毒力基因tir. stx2b和stx1b,运用原核表达系统在体外进行串联表达,获得重组蛋白。选择能够增强肠通透的霍乱弧菌封闭小带毒素zot基因进行串联,希望能够增强机体的粘膜免疫,尤其是肠道的粘膜免疫。选用链霉素处理的Balb/c小鼠作为评价动物,皮下和鼻腔两个途径免疫Stx2B-Tir-Stx1B-Zot蛋白,通过测定血清中的IgG滴度和IgA滴度、粪便中的IgA滴度以及粪便排菌来衡量Stx2B-Tir-Stx1B-Zot蛋白的免疫原性,为EHEC O157:H7亚单位疫苗的研制筛选良好的免疫原。
1.肠出血性大肠杆菌0157：H7 tir基因重组蛋白的表达及鉴定
克隆、表达肠出血性大肠杆菌(enterohaemorrhagic Escherichia coli, EHEC)0157：H7转位紧密素受体(tir)基因,并研究其免疫原性。选用pET28原核表达载体,构建pET28-tir重组质粒,并在大肠杆菌BL21中进行高效表达。选用家兔制备高滴度多克隆抗体,Western blot分析其免疫原性。结果显示成功获得高效表达的重组Tir蛋白,并制备了兔源Tir多克隆抗体,Western blot分析此抗体能与EHEC O157:H7(?)勺天然Tir蛋白发生特异性抗原抗体反应。我们在大肠杆菌中成功克隆表达了tir基因,所获重组Tir蛋白具有良好的反应原性。
2.大肠杆菌O157:H7 Tir重组蛋白的生物学特性
以HEp-2细胞为模型,评价重组Tir蛋白制备的抗体是否能够抑制(?)EHEC O157:H7的粘附和A/E损伤的形成。应用纯化后的重组Tir蛋白,皮下免疫Balb/c小鼠进行免疫保护性实验。两免断尾采血,间接ELISA测定血清特异性抗体效价。二免14d,10’。CFUEHEC O157:H7口服灌胃攻毒。每隔两天采集粪便检测排菌量。结果显示：表达的Tir蛋白具有天然Tir蛋白的功能特性,其抗体能够较大程度地抑制EHEC 0157：H7对HEp-2细胞的粘附和A/E损伤的形成。二免Balb/c小鼠存活率高达100%,排菌减少,8d后检测不到排菌,而对照组在第16d仍然能检测到排菌。因此,体外表达的重组Tir蛋白具有良好的抗原性,可作为EHEC O157:H7基因工程疫苗研制的首选抗原之一
3.肠出血性大肠杆菌O157:H7 Stx2B-Tir-StxlB-Zot多价融合蛋白表达及鉴定
EHEC O157:H7主要定植在肠道内,研制能够增强粘膜免疫基因工程疫苗具有很大优势。构建表达tir和stxb的融合基因,将tir基因中间295个氨基酸残基(Tir295)与stXB亚基基因72个氨基酸串联构建ppGEX-stx2b-tir-stx1b重组质粒,并与霍乱弧菌封闭小带毒素(Zot)靠近C端基因串联,构建重组质粒pGEX-stx2b-tir-stxl b-zot,转化于BL21(DE3),用IPTG进行诱导表达,经SDS-PAGE电泳检测,该融合蛋白获得了高效表达,主要以可溶性形式存在于菌体胞浆中。Western blot鉴定显示,Stx2B-Tir-Stx1B-Zot具有良好的反应原性。
4.大肠杆菌O157:H7 Stx2B-Tir-StxlB-Zot重组蛋白的免疫原性
Stx2B-Tir-Stx1B-Zot重组蛋白,分别皮下和鼻腔免疫Balb/c小鼠,二次免疫后测定血清中IgG和IgA滴度,粪便中IgA滴度。定期采集粪便,测定粪便排菌情况。结果显示：鼻腔免疫后Balb/c小鼠存活率100%,攻毒7d后检测不到排菌,并且最高排菌量只有3×103CFU/0.1g,血清中IgG滴度接近105,粪便中IgA接近102；皮下免疫后Balb/c小鼠存活率88%,攻毒9d后检测不到排菌,并且最高排菌量只有1.1×105CFU/0.1g,血清中IgG滴度也接近105,粪便中检测不到IgAStx2B-Tir-Stx1B-Zot鼻腔免疫能够发挥更好的免疫原性,可以作为EHEC 0157：H7亚单位疫苗研制的免疫原。
Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic enteric pathogen of worldwide importance, and is one of Shiga toxin-producing Escherichia coli (STEC). The type III secretion system of E. coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E.coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXcp of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery.
In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-StxlB-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST'Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Our aim is to determine whether antibodies specific for Stx2B-Tir-Stx1B-Zot could prevent O157:H7 colonization, to determine the role of Zot in the blocking colonization as well as to develop the subunit vaccine for EHEC O157:H7.
1. Construction and Expression of Recombinant Protein Tir from Escherichia coli O157:H7
The objective of the experiment is to clone, express and study the antigenicity of translocation intimin receptor(tir) gene from EHEC O157:H7. The vector pET28 and BL21(DE3) were used to construct and overexpress the recombinant protein of tir gene by prokaryotic expression. The recombinant Tir protein was used to immunize rabbits to obtain high-titer polyclonal antibodies,which was used to analyze the antigenicity of Tir by Western blot. We obtain successfully high-level expression of recombinant Tir protein and the molecular weight is 48kD.The polyclonal antibody against Tir protein have good a reactive fragment with the native Tir from Escherichia coli O157:H7. In conclusion, tir gene was cloned and expressed successfully in Escherichia coli, and recombinant Tir protein has a good antigenicity.
2. Biological Characterization of Recombinant Tir Protein from Escherichia coli O157:H7
HEp-2 cell and the polyclonal anti-Tir were used to evaluate adherence, adherence inhibition and attaching and effacing lesion (A/E) of EHEC O157:H7. Balb/c mice were inoculated with purified 100μg Tir protein subcutaneously and the protection rate and fecal shedding were analyzed after orally challenged by 1010CFU EHEC O157:H7. Anti-Tir IgG titers were detected by indirect ELISA. Balb/c mice immunized twice showed survival rate up to 100% compared to the 50% survival rate. Moreover, no EHEC O157:H7 in the fecal samples was detected after 8d challenged with O157:H7,but non-immunized mice still sheded the organism on 16th day.The above-mentioned results indicated that Tir protein has a good antigenicity and could be used for EHEC 0157 genetic engineering vaccines.
3. Expression Stx2B-Tir-StxlB-Zot Multivalent fusion protein from EHEC O157:H7
EHEC O157:H7 mainly colonize the intestine tissue, therefore, developing the genetic engineering vaccine enhancing the mucosal immunity has a significant dominance. In this study, we amplified tir, stx1b and stx2b genes and contructed the recombinant plasmid of pGEX-stx2b-tir-stx1b. The positive pGEX-stx2b-tir-stx1b were digested with Xho I, dephosphorylated with alkaline phosphatase and ligated with the zot gene to construct the pGEX-stx2b-tir-stx1b-zot. BL21(pGEX-stx2b-tir-stx1b-zot) was induced with IPTG and analysize with SDS-PAGE. All the results indicated that the recombinant Stx2B-Tir-Stx1B-Zot was successfully expressed, the molecular weight is 107kD and the anti-Stx2B-Tir-Stx1B-Zot antibody are able to react with the native Stx2B,Tir and Stx1B from EHEC 0157:H7.
4. Immunogenicity Analysis of Stx2B-Tir-StxlB-Zot from EHEC O157:H7
Balb/c mice were immunized subcutaneously and intranasally with Stx2B-Tir-StxlB-Zot protein, respectively at week 0,3. After the second immunization, the blood from the immunized and naive mice were collected to prepare sera for detecting the IgG and IgA titers. The feces form the immunized and naive mice were collected to detect the secretory IgA titers. Balb/c mice immunized intranasally have 100% survival rate, no detectable bacteia on the 7th after challenged, the highest shedding of 3×103CFU/0.1g and 105 IgG titers in sera and aroud102IgA titers in feces. Balb/c mice immunized subcutanously have 88% survival rate, no detectable bacteia on the 9th after challenged, the highest shedding of 1.1×105CFU/0.1g and 105 IgG titers in sera and no IgA titers in feces. Stx2B-Tir-Stx1B-Zot immunized intranasally is capable to play better immogenicity and able to be a preferred antigen to develop the subunit vaccine for EHEC O157:H7.
引文
[1]Riley LW, Remis RS, Helgerson SD,et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype [J]. The New England Journal of Medicine,1983,308(12):381-385.
[2]Wells JG, Davis B, Wachsmuth JK,et al. Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype [J] Journal of Clinical Microbiology,1983,8(3):51 2-520.
[3]Whittam TS, Wolfe ML,Wachsmuth IK,et al. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea [J].Infection and Immunity,1 993,61(5):1619-1629.
[4]Reid SD, Herbelin CJ,Bumbaugh AC,et al. Parallel evolution of virulence in pathogenic Escherichia coli [J]. Nature,2000,406:64-67.
[5]Shaikh N,Tarr P. Escherichia coli O 157:H7 Shiga toxin-encoding bacteriophages:integrations, excisions,truncations, and evolutionary implications[J] Journal of Bacteriology,2003,1 85(2):3596-3605.
[6]Perna NT,Plunkett G,Burland V,et al. Genome sequence of enterohaemorrhagic Escherichia coli O1 57:H7[J].Nature,2001,409:529-533.
[7]Bako W. Enterohemorrhagic Escherichia coli 0157 infection in children [J].Med Sci Monit,2003,9 (4):14-18.
[8]Paton JC, Paton,AW. Shiga toxigenic Escherichia coli infections [J].Science&Medicine,2007 (3):28-37.
[9]汪华,景怀琦,李红卫,等.江苏省淮北地区肠出血性大肠埃希菌O157：H7感染性腹泻并发急性肾衰的研究[J].中华流行病学杂志,2004,25(11)：938-940.
[10]Koyange L.Enterohemorrhagic Escherichia coli O157,Kinshasa [J].Emerg Infect Dis,2004,10 (5):968-969.
[11]Karch H. Sorbitol-Fermenting Shiga toxin2 producing Escherichia coli O157:H-strains: epidemiology,phenotypic and molecular characteristics, and microbiological diagnosis [J]. J Clin Mcrobieol,2001,39 (6):2043-2049.
[12]Perna NT, Plunkett G,Burland V,et al.Genome sequence of enterohaemorrhagic Escherichia coli O157:H7[J].Nature,2001,409:529-533.
[13]李洪卫,逢波,景怀琦.徐州市2002年肠出血性大肠埃希菌O157：H7感染性腹泻的调查.中华流行病学杂志[J],2002,23(2)：119-122.
[14]汪华.肠出血性大肠杆菌O157：H7流行特征和控制对策研究.医学研究通讯[J],2005,34(5)：24-25.
[15]Beuchat LR. Pathogenic microorganisms associated with fresh produce[J].Journal of Food Protection.1996,59:204-216.
[16]Tauxe RH, Kruse C,Hedberg M. Microbial hazards and emerging issues associzted with produce. A preliminary report to the National Advisory Committee on Microbiological Criteria for Foods [J]. Journal of Food Protection.1997,60:1400-1408.
[17]Rowe PC,Orrbine E,Ogborn M,et al.Epidemic Escherichia coli O157:H7 gastroenteritis and hemolytic-uremic syndrome in a Canadian Inuit community:intestinal illness in family members as a risk factor.J.Pediatr.1994,124:21-26
[18]Belongia EA, Osterholm MT,Soler JT,et al. Transmission of Escherichia coli 0157:H7 infection in Minnesota child day-care facilities[J].The Journal of the American Medical Association,1993, 269(7):883-888.
[19]Chalmers RM, Aird H,Bolton FJ.Waterborne Escherichia coli O157[J]. Journal of Applied Microbiology,2000,88:124-132.
[20]Verma A, BoRon FJ, Fiefield D,et al. An outbreak of E.coli O157 associated with a swimming pool:an unusual vehicle of transmission[J].Epidemiology and lnfecttion,2007,135(6):989-992.
[21]Armstrong GL, Hollingsworth J,Morris Jr J G.Emerging foodborne pathogens:Escherichia coli O157:H7:a model of entry of a new pathogen into the food supply of the developed world [J].EpidemiologicReviews,1996,1(80):20-25.
[22]Ogden D, Hepburn NF, Macrae M,et al. Long-term survival of Escherichia coli 0157 on pasture following all outbreak aSsociated with sheep at a scout camp [J].Letters in Applied Microbiology,2002,34(2):100-104.
[23]Nataro JP, Kaper JB.Diarrheagenic Escherichia coli [J].Clinical Microbiological Reviews,1998,1 (1):142-201.
[24]LuoY,Frey E, Pfuetzner,et al. Crystal structure of enteropathogenic Escherichia coli Intimin-receptor complex [J].Nature,2000,405(29):1073-1077.
[25]Li YL, Frey E, Mackenzie AM, et al. Human response to Escherichia coli 0157:H7 infection: antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9):5090-5095.
[26]Cornick NA, Helgerson AF, Sharma V. Shiga toxin and Shiga toxin-encoding phage do not facilitate Escherichia coli O157:H7 colonization in sheep [J]. Appl Environ Microbiol, 2007;73:344-346.
[27]Robinson CM, Sinclair JF, Smith MJ, et al. Shiga toxin of enterohemorrhagic Escherichia coli type O157:H7 promotes intestinal colonization [J]. Proc Natl Acad Sci USA,2006,103:9667-9672.
[28]Menge C, Wieler LH, Schlapp T, Baljer G. Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro [J]. Infect Immun,1999, 67:2209-2217.
[29]Schuller S, Heuschkel R, Torrente F, et al. Shiga toxin binding in normal and inflamed human intestinal mucosa[J]. Microb Infect,2007,9:35-39.
[30]Smith DG, Naylor SW, Gally DL. Consequences of EHEC colonisation in humans and cattle [J]. Int J Med Microbiol,2002,292:169-83.
[31]Yin X, Chambers JR, Wheatcroft R, et al. Adherence of Escherichia coli O157:H7 mutants in vitro and in ligated pig intestines [J]. Appl Environ Microb 2009;75(15):4975-83.
[32]Liu B, Yin X, Feng Y, et al. Verotoxin 2 enhances adherence of enterohemorrhagic Escherichia coli O157:H7 to intestinal epithelial cells and expression of β1-integrin by IPEC-J2 Cells [J]. Appl Environ Microb,2010;76(13):4461-4468.
[33]Johannes Muthing, Christian H,Schweppe, et al. Shiga toxins, glycosphingolipid diversity, and endothelial cell injury. Thromb Haemost[J],2009,101(2):252-264.
[34]Van Donkersgoed J,Hancock D,Rogean D.etal.Escherichia coli O157:H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan[J].Can Vet J,2005,46(8):724-728.
[35]Wen SX,Teel LD,Judge NA,e t al. Genetic toxoids of Shigatoxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge [J].Vaccine,2006,2024(8):1142-1148.
[36]易勇,邹全明,程建平,等.Stx2B与IntiminC300融合蛋白的构建、表达及免疫保护研究[J].中华微生物学和免疫学杂志,2005,25(3)：227-232.
[37]程建平,邹全明,毛旭虎,等.肠出血性大肠杆菌O157：H7感染动物模型的建立[J].中国人兽共患病杂志,2005,21(4)：276-279.
[38]Boyd B, Richardson S and Gariepy J. Serological, responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin [J]. Infect Immun,1991,59(3):750-757.
[39]Ishikawa S, Kawahara K, Kagami Y, et al. Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin, InfectImmun,2003,71(6):3235-3239.
[40]Konadu E,Donohue-Rolfe A,Calderwood SB,et al.Syntheses and immunologic properties of Escherichia coli 0157 O-specific polysaccharide and Shiga toxin 1B subunit conjugates in mice[J] Infect Immun,1999,67(11):6191-6193.
[41]Ahmed A, Li J, Shiloach Y, et al. Safety and immunogenicity of Escherichia coli 0157 O-specific polysacchare conjugate vaccine in 2-5-year-old children [J]. Infect Dis,2006,193(4):515-521.
[42]Butterton JR, Ryan ET, Acheson DW, et al. Coexpression of the B subunit of Shiga toxin 1 and EaeA from enterohemorrhagic Escherichia coli in Vibriocholerae vaccine strain[J]. Infect Immun, 1997,65:2127-2135.
[43]Yamasaki S. Development of vaccine for enterohemorrhagic Escherichia coli infection [J]. Nippon Rinsho,2002,60(6):1083-1088.
[44]Mayr UB,Hailer C,Haidinger W,et al.Bacterial Ghosts as an Oral Vaccine, a Single Dose of Escherichia coli O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge[J].Infection and Immunity,2005,73(8):4810-4817.
[45]Sinclair JF, O'Brien AD. Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7 [J]. J Biol Chem,2002, 277:2876-2885.
[46]Fasano AC, Fiorentini GD, Uzzau S,et al. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro[J]. J. Clin. Investig,1995,96:710-720.
[47]Baudry B,Fasano A,Ketley J,et al.Cloning of gene(zot)encoding a new toxin produced by Vibrio cholerae [J].Infection and Immunity,1992,60(3):428-434.
[48]何志勇,陈哲宇,王东宁,等.霍乱弧菌zot基因的克隆及其在大肠杆菌中的表达[J].生物工程学报,2000,16(5)：570-573.
[49]Mariarosaria Di Pierro, Ruliang Lu, Sergio Uzzau, et al. Zonula Occludens Toxin Structure-Function Analysis [J]. J. Bio Chem.2001,276 (22):19160-19165.
[50]Mariarosaria Marinaro, Alessio Fasano, Maria Teresa De Magistrisl. Zonula Occludens Toxin Acts as an Adjuvant through Different Mucosal Routes and Induces Protective Immune Responses[J]. Infect Immun.2003,71(4):1897-1902.
[51]Mauro R,Francesco M,Diomira L,et al. Zonula occludens toxin(zot) interferes with the induction of nasal tolerance to gliadian[J].Immunology Letters,2002,81(2):217-221.
[52]Fasano A,Fiorentini C,Donelli G,et al.Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization in vitro [J].clinieal Investigation,1995,96(6):710-720.
[53]Cox D, Gao H, Raje S, et al.Enhancing the permeation of marker compounds and enaminone anticonvulsants across Caco-2 monolayers by modulating tight junctions using zonula occludens toxin[J].Eur J Pharm Biopharm,2001,52(2):145-150.
[54]Uzzau S, Lu R, Wang W, Fiore C, Fasano A. Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo-2) and murine (IEC6) intestinal cell lines[J]. FEMS Microbiol. Lett 2001,194:1-5.
[55]胡轶鹏.封闭带毒素的融合表达[D]广州：华南农业大学,2003.
[56]王静.封闭带毒索功能片段△G的原核表达及活性研究[D].广州：华南农业大学,2004.
[57]Fasano A.Innovative strategies for the oral delivery of drugs and peptides[J].Tibteeh Apprll,1998,16:152.
[1]Centers for Disease Control and Prevention (CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--10 states,2008 [J]. Morbidity and Mortality Weekly Report.2009,57(14):366-370
[2]Peter Feng. Escherichia coli Serotype O157:H7:Novel Vehicles of Infection and Emergence of Phenotypic Variants[J]. Emerging Infectious Diseases,2005, 1(2),47-52.
[3]Doyle MP, Erickson MC. Summer meeting 2007-the problems with fresh produce:an overview[J]. Journal Applied Microbiology.2008,105(2):317-30.
[4]Li Y, Frey E, Mackenzie,et al. Human response to Escherichia coli 0157:H7 infection:antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9):5090-5095.
[5]Potter AA, Klashinsky S, Li Y, et al. Decreased shedding of. Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins[J]. Vaccine,2004,22:362-369.
[6]Yuhng L, Elizabeth F, Andrew M, et al. Human response to Escherichia coli O157:H7 infection 1 antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9)15090-15095.
[7]Jiu H, Magoun L, Luperehio S, et al.The Tir-binding region of enterohemorrhagic Escherichia coli intimin is sufficient to trigger aaron condensation after bacterial-induced host cell signaling[J]. Molecular Microbiology,1999,34(1):67-81.
[8]McKee ML, Melton-Celsa AR, Moxley RA, et al. Escherichia coli 0157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells[J]. Infection Immunity, 1995,63:3739-3744
[9]Torres AG and Kaper JB. Multiple elements controlling adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa cells[J]. Infection Immunity,2003,71:4985-4995
[10]Naylor SW, Roe AJ, Nart P, et al. Escherichia coli O157:H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon[J]. Microbiology 2005,151:2773-81.
[11]Hartland EL, Batehelor M, Delahay RM, et al. Binding of intimin from enteropathogic Escherichia coli to Tir and to host cells[J]. Molecular Microbiology,1999,32:151-158.
[12]Kuhne SA, Hawes WS, La Ragione RM, et al. Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7[J]. Journal Clinical Microbiology,2004,42(7):2966-2976.
[13]Amani J, Mousavi SL, Rafati S, et al. In silico analysis of chimeric espA,eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications[J].Theoretical Biology Medical Model,2009,6:28.
[1]Centers for Disease Control and Prevention (CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--10 states,2008 [P]. Morbidity and Mortality Weekly Report,2009,57(14):366-370
[2]Feng P. Escherichia coli Serotype 0157:H7:novel vehicles of infection and emergence of phenotypic variants[J]. Emerging Infectious Diseases,2005,1(2),47-52.
[3]Doyle MP, Erickson MC. Summer meeting 2007-the problems with fresh produce:an overview[J]. Journal Applied Microbiology,2008,105(2):317-30.
[4]Li YL, Frey E, Mackenzie AR, et al. Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9):5090-5095.
[5]Potter AA, Klashinsky S, Li Y, et al. Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type Ⅲ secreted proteins[J]. Vaccine,2004,22:362-369.
[6]Fujii J,Kinoshita Y,Yamada Y,et al.. Neurotoxicity of intrathecal Shiga toxin2 and protection by intrathecal injection of anti-Shiga toxin 2 antiserum in rabbits[J]. Microbial Pathogenesis,1998,25(3):139-146.
[7]Shawn Babiuk, David JA, Dragan Rogan, et al. Subcutaneous and intranasal immunization with type Ⅲ secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice[J]. Microbial Pathogenesis,2008:45:7-11.
[8]McKee ML, Melton-Celsa AR, Moxley RA, et al. Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells[J]. Infection Immunity,1995,63: 3739-3744.
[9]Torres A G, Kaper J B. Multiple elements controlling adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa cells[J]. Infection Immunity,2003,71:4985-4995.
[10]Caparon M G, Stretptococci D S, Arne Oisen. Role of M protein in adherence of group A stretptocci[J]. Microbial Pathogenesis,1992,12:199-208.
[11]顾天钊,陆承平,陈怀青.鲍氏不动杆菌MF1株的黏附特性,南京农业大学学报,1999,22(3)：65-68.
[12]Knutton S, Baldwin T, Williams PH, et al.Actin accumulation at sites of bacterial adhesion to tissue culture cells:basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli[J]. Infection Immunity,1989,57:1290-1298.
[13]Dean-Nystrom EA, Gansheroff LJ, Mills M, et al. Vaccination of pregnant dams with intimin (0157) protects suckling piglets from Escherichia coli O157:H7 infection[J]. Infection Immunity, 2002,70:2414-2418.
[14]Babiuk S, Asper DJ, Rogan D, et al. Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice[J]. Microbial Pathogenesis,2008,45:7-11.
[15]Judge NA, Mason HS, O'Brien AD. Plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of Escherichia coli 0157:H7 shedding in feces[J]. Infection Immunity, 2004,72(1):168-175
[16]Chase TM, Gally D, Low C, et al. Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157[J]. National Review Microbiology,2008,6:904-912.
[17]Yoon JW, Hovde CJ. All blood, No stool:enterohemorrhagic Escherichia coli 0157:H7 infection[J]. Journal Veterinary Sciences,2008; 9:219-231.
[18]Tarr PI, Gordon CA, Chandler W. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome[J]. Lancet,2005,365:1073-1086.
[19]Tesh V L. Shiga toxins not just cytotoxins anymore[J]. Trends Microbiology,2001,9:584-585.
[20]Naylor SW, Low JC, Besser TE, et al. Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli 0157:H7 in the bovine host[J]. Infection Immunity,2003,71:1505-1512.
[21]Naylor SW, Nart P, Sales J, et al. Impact of the direct application of therapeutic agents to the terminal recta of experimentally colonized calves on Escherichia coli O157:H7 shedding[J]. Applied Environment Microbiology,2007,73:1493-1500.
[22]Meng J, Zhao S, Doyle M P. Virulence genes of Shiga toxin-producing Escherichia coli isolated from food, animals and humans[J]. International Journal Food Microbiology,1998,45:22-25.
[23]Ebel F, Podzadel T, Rohde M,et al. Initial binding of Shiga toxin-producing Eschedchia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA containing surface appendage[J]. Molecular Microbiology,1998,30(1):147-161.
[24]Hartland E L, Batehelor M, Delahay R M. Binding of intimin from enteropathogic Escherichia coli to Tir and to host cells[J]. Molecular Microbiology,1999,32(1):151-158.
[1]Centers for Disease Control and Prevention (CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--10 states,2007[P]. MMWR Morbity Mortal Wkly Report.2008,57(14):366-370.
[2]Reinstein S, Fox JT, Shi X, et al, Nagaraja TG. Prevalence of Escherichia coli O157:H7 in the American bison (Bison bison) [J]. Journal of Food Protection,2007,70(11):2555-2560.
[3]Doyle MP, Erickson MC. Summer meeting 2007-the problems with fresh produce:an overview[J]. Journal of Applied Microbiology,2008,105:317-330.
[4]Marcus R. New information about pediatric foodborne infections:the view from FoodNet[J]. Current Opinion Pediatriation,2008,20(1):79-84.
[5]Jiu H, Magoun L, Luperehio S, et al.The Tir-binding region of enterohemorrhagic Escherichia coli intimin is suficient to trigger aaron condensation after bacterial-induced host cell signaling[J]. Mol ecular Microbiology,1999,34(1):67-81.
[6]Li Y, Frey E, Mackenzie AM,et al. Human response to Escherichia coli O157:H7 infection:antibodies to secreted virulence factors[J].Infection Immunity,2000,68(9):5090-5095.
[7]Xianhua Yin, James R. Chambers, et al. Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines[J]. Applied Environmental Microbiology,2009,75(15):4975-4983.
[8]Bianfang Liu, Xianhua Yin, Yanni Feng, et al. Verotoxin 2 Enhances Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Intestinal Epithelial Cells and Expression of Pl-Integrin by IPEC-J2 Cells[J]. Applied Environmental Microbiology,2010,76(13):4461-4468.
[9]Xiang Gao, Kun Cai, Jing Shi, et al. Immunogenicity of a novel Stx2B-Stx1B fusion protein in a mice model of Enterohemorrhagic Escherichia coli O157:H7 infection[J]. Vaccine, 2009,27:2070-2076.
[10]Gyles CL. Shiga toxin-producing Escherichia coli:An overview. Journal of Animal Science, 2007.85:E45-E62.
[11]Xuehan Zhang, Kongwang He, MAO Ai-hua, ZHOU-Jun ming, YU Zheng-yu, WEN Li-bin, NI Yan-xiu, GUO Rong-li, LU Li-xin. Cloning, Expression, Biological Activities of Recombinant Tir gene from Enterohemorrhagic Escherichia coli O157:H7[J]. Scientia Agricultura Sinica. 2010,43(12):2570-2577.
[12]Fujii J,Kita T,Yoshida S, et al. Direct evidence of neuron impairment by oral infection with verotoxin-producing Escherichia coli O157:H7 in mitomycin-treated mice[J]. Infection Immunity,1994,62 (8):3447-3453.
[13]陈萍,冯书章,孙洋,等.出血性大肠杆菌0157 Eae/Stxl/2B融合蛋白的免疫学特性[J].中国兽医学报,2007,27(6)：838-841.
[14]张雪寒,何孔旺,卢维彩,等.出血性大肠杆菌O157：H7的Tir与stxl/2B融合基因的构建和表达[J].内蒙古农业科技,2009(3)：59-61.
[1]Deibel C, Kramer S, Chakraborty T, et al. a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine-phosphorylated 90 kDa protein[J]. Molecular Microbiology,1998,28:463-474.
[2]Ray PE, Liu XH. Pathogenesis of Shiga toxin-induced hemolytic uremic syndrome [J]. Pediatration. Nephrology,2001,16:823-839.
[3]Babiuk S, Asper DJ, Rogan D,et al. Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice[J]. Microbial Pathog enesis,2008,45:7-11.
[4]Wadolkowski EA,Burris JA, O'Brien AD. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7[J]. Infection Immunity,1990,58:2438-45.
[5]Mariarosaria Di Pierro, Ruliang Lu, Sergio Uzzau, et al. Zonula Occludens Toxin Structure-Function Analysis[J]. Journal of BioChemistry,2001,276 (22):19160-19165.
[6]Maria Teresa De Magistris. Zonula occludens toxin as a new promising adjuvant for mucosal vaccines[J]. Vaccine.2006,24S2:S2/60-S2/61.
[7]Fasano AC, Bernadette Baudry, Pumplin DW, et al. Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions [J]. Proceeding Nattional Academy Science USA,1991,88: 5242-5246.
[8]van Ginkel FW, Jackson RJ, Yuki Y, et al. The mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues[J]. Journal of Immunology,2000,165:4778-4782.
[9]Mariarosaria Marinaro, Alessio Fasano, Maria Teresa De Magistris 1. Zonula Occludens Toxin Acts as an Adjuvant through Different Mucosal Routes and Induces Protective Immune Responses[J]. Infection Immunity,2003,71(4):1897-1902.
[10]Marinaro M, Di Tommaso A, Uzzau S, et al. Zonula occludens toxin is a powerful mucosal adjuvant for intranasally delivered antigens[J]. Infection Immunity,1999,67:1287-1291.
[11]Fasano A. Regulation of intercellular tight junctions by Zonula occludens toxin and its eukaryotic analogue zonulin[J]. Annals of the New York Academy Science,2000,915:214-222.
[2]Wells JG, Davis B, Wachsmuth JK,et al. Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype [J] Journal of Clinical Microbiology,1983,8(3):51 2-520.
[3]Whittam TS, Wolfe ML,Wachsmuth IK,et al. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea [J].Infection and Immunity,1 993,61(5):1619-1629.
[4]Reid SD, Herbelin CJ,Bumbaugh AC,et al. Parallel evolution of virulence in pathogenic Escherichia coli [J]. Nature,2000,406:64-67.
[5]Shaikh N,Tarr P. Escherichia coli O 157:H7 Shiga toxin-encoding bacteriophages:integrations, excisions,truncations, and evolutionary implications[J] Journal of Bacteriology,2003,1 85(2):3596-3605.
[6]Perna NT,Plunkett G,Burland V,et al. Genome sequence of enterohaemorrhagic Escherichia coli O1 57:H7[J].Nature,2001,409:529-533.
[7]Bako W. Enterohemorrhagic Escherichia coli 0157 infection in children [J].Med Sci Monit,2003,9 (4):14-18.
[8]Paton JC, Paton,AW. Shiga toxigenic Escherichia coli infections [J].Science&Medicine,2007 (3):28-37.
[9]汪华,景怀琦,李红卫,等.江苏省淮北地区肠出血性大肠埃希菌O157：H7感染性腹泻并发急性肾衰的研究[J].中华流行病学杂志,2004,25(11)：938-940.
[10]Koyange L.Enterohemorrhagic Escherichia coli O157,Kinshasa [J].Emerg Infect Dis,2004,10 (5):968-969.
[11]Karch H. Sorbitol-Fermenting Shiga toxin2 producing Escherichia coli O157:H-strains: epidemiology,phenotypic and molecular characteristics, and microbiological diagnosis [J]. J Clin Mcrobieol,2001,39 (6):2043-2049.
[12]Perna NT, Plunkett G,Burland V,et al.Genome sequence of enterohaemorrhagic Escherichia coli O157:H7[J].Nature,2001,409:529-533.
[13]李洪卫,逢波,景怀琦.徐州市2002年肠出血性大肠埃希菌O157：H7感染性腹泻的调查.中华流行病学杂志[J],2002,23(2)：119-122.
[14]汪华.肠出血性大肠杆菌O157：H7流行特征和控制对策研究.医学研究通讯[J],2005,34(5)：24-25.
[15]Beuchat LR. Pathogenic microorganisms associated with fresh produce[J].Journal of Food Protection.1996,59:204-216.
[16]Tauxe RH, Kruse C,Hedberg M. Microbial hazards and emerging issues associzted with produce. A preliminary report to the National Advisory Committee on Microbiological Criteria for Foods [J]. Journal of Food Protection.1997,60:1400-1408.
[17]Rowe PC,Orrbine E,Ogborn M,et al.Epidemic Escherichia coli O157:H7 gastroenteritis and hemolytic-uremic syndrome in a Canadian Inuit community:intestinal illness in family members as a risk factor.J.Pediatr.1994,124:21-26
[18]Belongia EA, Osterholm MT,Soler JT,et al. Transmission of Escherichia coli 0157:H7 infection in Minnesota child day-care facilities[J].The Journal of the American Medical Association,1993, 269(7):883-888.
[19]Chalmers RM, Aird H,Bolton FJ.Waterborne Escherichia coli O157[J]. Journal of Applied Microbiology,2000,88:124-132.
[20]Verma A, BoRon FJ, Fiefield D,et al. An outbreak of E.coli O157 associated with a swimming pool:an unusual vehicle of transmission[J].Epidemiology and lnfecttion,2007,135(6):989-992.
[21]Armstrong GL, Hollingsworth J,Morris Jr J G.Emerging foodborne pathogens:Escherichia coli O157:H7:a model of entry of a new pathogen into the food supply of the developed world [J].EpidemiologicReviews,1996,1(80):20-25.
[22]Ogden D, Hepburn NF, Macrae M,et al. Long-term survival of Escherichia coli 0157 on pasture following all outbreak aSsociated with sheep at a scout camp [J].Letters in Applied Microbiology,2002,34(2):100-104.
[23]Nataro JP, Kaper JB.Diarrheagenic Escherichia coli [J].Clinical Microbiological Reviews,1998,1 (1):142-201.
[24]LuoY,Frey E, Pfuetzner,et al. Crystal structure of enteropathogenic Escherichia coli Intimin-receptor complex [J].Nature,2000,405(29):1073-1077.
[25]Li YL, Frey E, Mackenzie AM, et al. Human response to Escherichia coli 0157:H7 infection: antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9):5090-5095.
[26]Cornick NA, Helgerson AF, Sharma V. Shiga toxin and Shiga toxin-encoding phage do not facilitate Escherichia coli O157:H7 colonization in sheep [J]. Appl Environ Microbiol, 2007;73:344-346.
[27]Robinson CM, Sinclair JF, Smith MJ, et al. Shiga toxin of enterohemorrhagic Escherichia coli type O157:H7 promotes intestinal colonization [J]. Proc Natl Acad Sci USA,2006,103:9667-9672.
[28]Menge C, Wieler LH, Schlapp T, Baljer G. Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro [J]. Infect Immun,1999, 67:2209-2217.
[29]Schuller S, Heuschkel R, Torrente F, et al. Shiga toxin binding in normal and inflamed human intestinal mucosa[J]. Microb Infect,2007,9:35-39.
[30]Smith DG, Naylor SW, Gally DL. Consequences of EHEC colonisation in humans and cattle [J]. Int J Med Microbiol,2002,292:169-83.
[31]Yin X, Chambers JR, Wheatcroft R, et al. Adherence of Escherichia coli O157:H7 mutants in vitro and in ligated pig intestines [J]. Appl Environ Microb 2009;75(15):4975-83.
[32]Liu B, Yin X, Feng Y, et al. Verotoxin 2 enhances adherence of enterohemorrhagic Escherichia coli O157:H7 to intestinal epithelial cells and expression of β1-integrin by IPEC-J2 Cells [J]. Appl Environ Microb,2010;76(13):4461-4468.
[33]Johannes Muthing, Christian H,Schweppe, et al. Shiga toxins, glycosphingolipid diversity, and endothelial cell injury. Thromb Haemost[J],2009,101(2):252-264.
[34]Van Donkersgoed J,Hancock D,Rogean D.etal.Escherichia coli O157:H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan[J].Can Vet J,2005,46(8):724-728.
[35]Wen SX,Teel LD,Judge NA,e t al. Genetic toxoids of Shigatoxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge [J].Vaccine,2006,2024(8):1142-1148.
[36]易勇,邹全明,程建平,等.Stx2B与IntiminC300融合蛋白的构建、表达及免疫保护研究[J].中华微生物学和免疫学杂志,2005,25(3)：227-232.
[37]程建平,邹全明,毛旭虎,等.肠出血性大肠杆菌O157：H7感染动物模型的建立[J].中国人兽共患病杂志,2005,21(4)：276-279.
[38]Boyd B, Richardson S and Gariepy J. Serological, responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin [J]. Infect Immun,1991,59(3):750-757.
[39]Ishikawa S, Kawahara K, Kagami Y, et al. Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin, InfectImmun,2003,71(6):3235-3239.
[40]Konadu E,Donohue-Rolfe A,Calderwood SB,et al.Syntheses and immunologic properties of Escherichia coli 0157 O-specific polysaccharide and Shiga toxin 1B subunit conjugates in mice[J] Infect Immun,1999,67(11):6191-6193.
[41]Ahmed A, Li J, Shiloach Y, et al. Safety and immunogenicity of Escherichia coli 0157 O-specific polysacchare conjugate vaccine in 2-5-year-old children [J]. Infect Dis,2006,193(4):515-521.
[42]Butterton JR, Ryan ET, Acheson DW, et al. Coexpression of the B subunit of Shiga toxin 1 and EaeA from enterohemorrhagic Escherichia coli in Vibriocholerae vaccine strain[J]. Infect Immun, 1997,65:2127-2135.
[43]Yamasaki S. Development of vaccine for enterohemorrhagic Escherichia coli infection [J]. Nippon Rinsho,2002,60(6):1083-1088.
[44]Mayr UB,Hailer C,Haidinger W,et al.Bacterial Ghosts as an Oral Vaccine, a Single Dose of Escherichia coli O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge[J].Infection and Immunity,2005,73(8):4810-4817.
[45]Sinclair JF, O'Brien AD. Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7 [J]. J Biol Chem,2002, 277:2876-2885.
[46]Fasano AC, Fiorentini GD, Uzzau S,et al. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro[J]. J. Clin. Investig,1995,96:710-720.
[47]Baudry B,Fasano A,Ketley J,et al.Cloning of gene(zot)encoding a new toxin produced by Vibrio cholerae [J].Infection and Immunity,1992,60(3):428-434.
[48]何志勇,陈哲宇,王东宁,等.霍乱弧菌zot基因的克隆及其在大肠杆菌中的表达[J].生物工程学报,2000,16(5)：570-573.
[49]Mariarosaria Di Pierro, Ruliang Lu, Sergio Uzzau, et al. Zonula Occludens Toxin Structure-Function Analysis [J]. J. Bio Chem.2001,276 (22):19160-19165.
[50]Mariarosaria Marinaro, Alessio Fasano, Maria Teresa De Magistrisl. Zonula Occludens Toxin Acts as an Adjuvant through Different Mucosal Routes and Induces Protective Immune Responses[J]. Infect Immun.2003,71(4):1897-1902.
[51]Mauro R,Francesco M,Diomira L,et al. Zonula occludens toxin(zot) interferes with the induction of nasal tolerance to gliadian[J].Immunology Letters,2002,81(2):217-221.
[52]Fasano A,Fiorentini C,Donelli G,et al.Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization in vitro [J].clinieal Investigation,1995,96(6):710-720.
[53]Cox D, Gao H, Raje S, et al.Enhancing the permeation of marker compounds and enaminone anticonvulsants across Caco-2 monolayers by modulating tight junctions using zonula occludens toxin[J].Eur J Pharm Biopharm,2001,52(2):145-150.
[54]Uzzau S, Lu R, Wang W, Fiore C, Fasano A. Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo-2) and murine (IEC6) intestinal cell lines[J]. FEMS Microbiol. Lett 2001,194:1-5.
[55]胡轶鹏.封闭带毒素的融合表达[D]广州：华南农业大学,2003.
[56]王静.封闭带毒索功能片段△G的原核表达及活性研究[D].广州：华南农业大学,2004.
[57]Fasano A.Innovative strategies for the oral delivery of drugs and peptides[J].Tibteeh Apprll,1998,16:152.
[1]Centers for Disease Control and Prevention (CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--10 states,2008 [J]. Morbidity and Mortality Weekly Report.2009,57(14):366-370
[2]Peter Feng. Escherichia coli Serotype O157:H7:Novel Vehicles of Infection and Emergence of Phenotypic Variants[J]. Emerging Infectious Diseases,2005, 1(2),47-52.
[3]Doyle MP, Erickson MC. Summer meeting 2007-the problems with fresh produce:an overview[J]. Journal Applied Microbiology.2008,105(2):317-30.
[4]Li Y, Frey E, Mackenzie,et al. Human response to Escherichia coli 0157:H7 infection:antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9):5090-5095.
[5]Potter AA, Klashinsky S, Li Y, et al. Decreased shedding of. Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins[J]. Vaccine,2004,22:362-369.
[6]Yuhng L, Elizabeth F, Andrew M, et al. Human response to Escherichia coli O157:H7 infection 1 antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9)15090-15095.
[7]Jiu H, Magoun L, Luperehio S, et al.The Tir-binding region of enterohemorrhagic Escherichia coli intimin is sufficient to trigger aaron condensation after bacterial-induced host cell signaling[J]. Molecular Microbiology,1999,34(1):67-81.
[8]McKee ML, Melton-Celsa AR, Moxley RA, et al. Escherichia coli 0157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells[J]. Infection Immunity, 1995,63:3739-3744
[9]Torres AG and Kaper JB. Multiple elements controlling adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa cells[J]. Infection Immunity,2003,71:4985-4995
[10]Naylor SW, Roe AJ, Nart P, et al. Escherichia coli O157:H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon[J]. Microbiology 2005,151:2773-81.
[11]Hartland EL, Batehelor M, Delahay RM, et al. Binding of intimin from enteropathogic Escherichia coli to Tir and to host cells[J]. Molecular Microbiology,1999,32:151-158.
[12]Kuhne SA, Hawes WS, La Ragione RM, et al. Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7[J]. Journal Clinical Microbiology,2004,42(7):2966-2976.
[13]Amani J, Mousavi SL, Rafati S, et al. In silico analysis of chimeric espA,eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications[J].Theoretical Biology Medical Model,2009,6:28.
[1]Centers for Disease Control and Prevention (CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--10 states,2008 [P]. Morbidity and Mortality Weekly Report,2009,57(14):366-370
[2]Feng P. Escherichia coli Serotype 0157:H7:novel vehicles of infection and emergence of phenotypic variants[J]. Emerging Infectious Diseases,2005,1(2),47-52.
[3]Doyle MP, Erickson MC. Summer meeting 2007-the problems with fresh produce:an overview[J]. Journal Applied Microbiology,2008,105(2):317-30.
[4]Li YL, Frey E, Mackenzie AR, et al. Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors[J]. Infection Immunity,2000,68(9):5090-5095.
[5]Potter AA, Klashinsky S, Li Y, et al. Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type Ⅲ secreted proteins[J]. Vaccine,2004,22:362-369.
[6]Fujii J,Kinoshita Y,Yamada Y,et al.. Neurotoxicity of intrathecal Shiga toxin2 and protection by intrathecal injection of anti-Shiga toxin 2 antiserum in rabbits[J]. Microbial Pathogenesis,1998,25(3):139-146.
[7]Shawn Babiuk, David JA, Dragan Rogan, et al. Subcutaneous and intranasal immunization with type Ⅲ secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice[J]. Microbial Pathogenesis,2008:45:7-11.
[8]McKee ML, Melton-Celsa AR, Moxley RA, et al. Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells[J]. Infection Immunity,1995,63: 3739-3744.
[9]Torres A G, Kaper J B. Multiple elements controlling adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa cells[J]. Infection Immunity,2003,71:4985-4995.
[10]Caparon M G, Stretptococci D S, Arne Oisen. Role of M protein in adherence of group A stretptocci[J]. Microbial Pathogenesis,1992,12:199-208.
[11]顾天钊,陆承平,陈怀青.鲍氏不动杆菌MF1株的黏附特性,南京农业大学学报,1999,22(3)：65-68.
[12]Knutton S, Baldwin T, Williams PH, et al.Actin accumulation at sites of bacterial adhesion to tissue culture cells:basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli[J]. Infection Immunity,1989,57:1290-1298.
[13]Dean-Nystrom EA, Gansheroff LJ, Mills M, et al. Vaccination of pregnant dams with intimin (0157) protects suckling piglets from Escherichia coli O157:H7 infection[J]. Infection Immunity, 2002,70:2414-2418.
[14]Babiuk S, Asper DJ, Rogan D, et al. Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice[J]. Microbial Pathogenesis,2008,45:7-11.
[15]Judge NA, Mason HS, O'Brien AD. Plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of Escherichia coli 0157:H7 shedding in feces[J]. Infection Immunity, 2004,72(1):168-175
[16]Chase TM, Gally D, Low C, et al. Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157[J]. National Review Microbiology,2008,6:904-912.
[17]Yoon JW, Hovde CJ. All blood, No stool:enterohemorrhagic Escherichia coli 0157:H7 infection[J]. Journal Veterinary Sciences,2008; 9:219-231.
[18]Tarr PI, Gordon CA, Chandler W. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome[J]. Lancet,2005,365:1073-1086.
[19]Tesh V L. Shiga toxins not just cytotoxins anymore[J]. Trends Microbiology,2001,9:584-585.
[20]Naylor SW, Low JC, Besser TE, et al. Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli 0157:H7 in the bovine host[J]. Infection Immunity,2003,71:1505-1512.
[21]Naylor SW, Nart P, Sales J, et al. Impact of the direct application of therapeutic agents to the terminal recta of experimentally colonized calves on Escherichia coli O157:H7 shedding[J]. Applied Environment Microbiology,2007,73:1493-1500.
[22]Meng J, Zhao S, Doyle M P. Virulence genes of Shiga toxin-producing Escherichia coli isolated from food, animals and humans[J]. International Journal Food Microbiology,1998,45:22-25.
[23]Ebel F, Podzadel T, Rohde M,et al. Initial binding of Shiga toxin-producing Eschedchia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA containing surface appendage[J]. Molecular Microbiology,1998,30(1):147-161.
[24]Hartland E L, Batehelor M, Delahay R M. Binding of intimin from enteropathogic Escherichia coli to Tir and to host cells[J]. Molecular Microbiology,1999,32(1):151-158.
[1]Centers for Disease Control and Prevention (CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--10 states,2007[P]. MMWR Morbity Mortal Wkly Report.2008,57(14):366-370.
[2]Reinstein S, Fox JT, Shi X, et al, Nagaraja TG. Prevalence of Escherichia coli O157:H7 in the American bison (Bison bison) [J]. Journal of Food Protection,2007,70(11):2555-2560.
[3]Doyle MP, Erickson MC. Summer meeting 2007-the problems with fresh produce:an overview[J]. Journal of Applied Microbiology,2008,105:317-330.
[4]Marcus R. New information about pediatric foodborne infections:the view from FoodNet[J]. Current Opinion Pediatriation,2008,20(1):79-84.
[5]Jiu H, Magoun L, Luperehio S, et al.The Tir-binding region of enterohemorrhagic Escherichia coli intimin is suficient to trigger aaron condensation after bacterial-induced host cell signaling[J]. Mol ecular Microbiology,1999,34(1):67-81.
[6]Li Y, Frey E, Mackenzie AM,et al. Human response to Escherichia coli O157:H7 infection:antibodies to secreted virulence factors[J].Infection Immunity,2000,68(9):5090-5095.
[7]Xianhua Yin, James R. Chambers, et al. Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines[J]. Applied Environmental Microbiology,2009,75(15):4975-4983.
[8]Bianfang Liu, Xianhua Yin, Yanni Feng, et al. Verotoxin 2 Enhances Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Intestinal Epithelial Cells and Expression of Pl-Integrin by IPEC-J2 Cells[J]. Applied Environmental Microbiology,2010,76(13):4461-4468.
[9]Xiang Gao, Kun Cai, Jing Shi, et al. Immunogenicity of a novel Stx2B-Stx1B fusion protein in a mice model of Enterohemorrhagic Escherichia coli O157:H7 infection[J]. Vaccine, 2009,27:2070-2076.
[10]Gyles CL. Shiga toxin-producing Escherichia coli:An overview. Journal of Animal Science, 2007.85:E45-E62.
[11]Xuehan Zhang, Kongwang He, MAO Ai-hua, ZHOU-Jun ming, YU Zheng-yu, WEN Li-bin, NI Yan-xiu, GUO Rong-li, LU Li-xin. Cloning, Expression, Biological Activities of Recombinant Tir gene from Enterohemorrhagic Escherichia coli O157:H7[J]. Scientia Agricultura Sinica. 2010,43(12):2570-2577.
[12]Fujii J,Kita T,Yoshida S, et al. Direct evidence of neuron impairment by oral infection with verotoxin-producing Escherichia coli O157:H7 in mitomycin-treated mice[J]. Infection Immunity,1994,62 (8):3447-3453.
[13]陈萍,冯书章,孙洋,等.出血性大肠杆菌0157 Eae/Stxl/2B融合蛋白的免疫学特性[J].中国兽医学报,2007,27(6)：838-841.
[14]张雪寒,何孔旺,卢维彩,等.出血性大肠杆菌O157：H7的Tir与stxl/2B融合基因的构建和表达[J].内蒙古农业科技,2009(3)：59-61.
[1]Deibel C, Kramer S, Chakraborty T, et al. a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine-phosphorylated 90 kDa protein[J]. Molecular Microbiology,1998,28:463-474.
[2]Ray PE, Liu XH. Pathogenesis of Shiga toxin-induced hemolytic uremic syndrome [J]. Pediatration. Nephrology,2001,16:823-839.
[3]Babiuk S, Asper DJ, Rogan D,et al. Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice[J]. Microbial Pathog enesis,2008,45:7-11.
[4]Wadolkowski EA,Burris JA, O'Brien AD. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7[J]. Infection Immunity,1990,58:2438-45.
[5]Mariarosaria Di Pierro, Ruliang Lu, Sergio Uzzau, et al. Zonula Occludens Toxin Structure-Function Analysis[J]. Journal of BioChemistry,2001,276 (22):19160-19165.
[6]Maria Teresa De Magistris. Zonula occludens toxin as a new promising adjuvant for mucosal vaccines[J]. Vaccine.2006,24S2:S2/60-S2/61.
[7]Fasano AC, Bernadette Baudry, Pumplin DW, et al. Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions [J]. Proceeding Nattional Academy Science USA,1991,88: 5242-5246.
[8]van Ginkel FW, Jackson RJ, Yuki Y, et al. The mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues[J]. Journal of Immunology,2000,165:4778-4782.
[9]Mariarosaria Marinaro, Alessio Fasano, Maria Teresa De Magistris 1. Zonula Occludens Toxin Acts as an Adjuvant through Different Mucosal Routes and Induces Protective Immune Responses[J]. Infection Immunity,2003,71(4):1897-1902.
[10]Marinaro M, Di Tommaso A, Uzzau S, et al. Zonula occludens toxin is a powerful mucosal adjuvant for intranasally delivered antigens[J]. Infection Immunity,1999,67:1287-1291.
[11]Fasano A. Regulation of intercellular tight junctions by Zonula occludens toxin and its eukaryotic analogue zonulin[J]. Annals of the New York Academy Science,2000,915:214-222.