大肠杆菌K88ac-ST Ⅱ融合基因在减毒鼠伤寒沙门氏菌中的表达与免疫
|
摘要
为了构建表达大肠杆菌菌毛蛋白K88ac和热稳定肠毒素STII融合基因的无抗性减毒鼠伤寒沙门氏菌疫苗株,并对其免疫效果进行研究。
根据GenBank中登录号为m29375的K88ac基因序列以及减毒鼠伤寒沙门氏菌原核表达载体pYA3334的多克隆位点设计特异性引物P1、P2,并在上下游引物的5’端分别引入NcoI和BamHI酶切位点,从大肠杆菌DE3(pET- K88ac-STⅡ)中通过聚合酶链式反应(PCR)扩增得到K88ac-STⅡ融合基因。回收纯化后将K88ac-STⅡ融合基因用T4DNA连接酶连于克隆载体pBS-T上,热击转化受体大肠杆菌Top10,蓝白斑筛选阳性菌落,酶切鉴定证明其中含有K88ac-STⅡ融合基因片段,再测序鉴定该融合基因序列的正确性,命名为pBS-K88ac-STⅡ。之后采用缺失腺苷酸环化酶基因(Δcya)、环腺苷酸受体蛋白基因(Δcrp)以及天冬氨酸β-半醛脱氢酶基因(Δasd)的鼠伤寒沙门氏菌(X4550)作为宿主,将编码K88ac-STII的融合基因切下插入Asd+组成型表达载体pYA3334中,鉴定后通过电转化引入宿主菌X4550中,再次酶切鉴定正确后,过夜培养进行SDS-PAGE和Western-Blot试验,检测其特异性蛋白表达情况。最后分别以重组菌株X4550(pYA3334-K88ac-STII)灌服小鼠作为试验组,以转化了空载体的菌株X4550(pYA3334)作为空载体对照组,同时设PBS空白对照组,分别对三个分组的小鼠进行免疫,采集血清检测其抗体效价,之后免疫小鼠用强毒株C83915(STⅡ﹢)攻击,观察结果。
本实验成功构建了表达K88ac-STII融合基因平衡致死的减毒鼠伤寒沙门氏重组菌株X4550(pYA3334-K88ac-STII),该重组菌株在33Kda处有特异性表达的蛋白条带,与预期分子量大小一致,而空载体对照菌X4550(pYA3334)在该处未有相应条带出现。Western-blot免疫印迹结果显示重组菌株X4550(pYA3334-K88ac-STII)表达的蛋白能够被K88ac阳性血清特异性结合,在相应位置出现特异性显色条带。这证明重组菌株能够表达K88ac-STII特异性融合蛋白,重组菌的体外稳定实验也证明其有良好的稳定性,从而保证了K88ac-STII抗原能够得以持续表达。而且试验小鼠灌喂重组减毒鼠伤寒沙门氏菌X4550 (pYA3334-K88ac-STII)后,能够产生特异性的抗体,而且对大肠杆菌强毒株具有一定的抵抗力。这为进一步研制相应的抗仔猪大肠杆菌病及相应的沙门氏菌病的双价活疫苗侯选株奠定了重要基础。
Object:To construct a live oral Salmonella typhimurium vaccine strain expressing K88ac-STII fusion gene of enterotoxigenic Escherichia coli.
Methods: Primers(P1,P2) were designed according to the nucleotide sequence of K88ac gene of enterotoxigenic Escherichia coli(ETEC) reported respectively and the expression vector pYA3334 . With the specific primers, the gene encoding the fusion protein K88ac-STII was amplified by PCR of the plasmid pET- K88ac-STⅡand was inserted into the linearized plasmid vector PBS-T. By enzyme analysis, PCR and sequencing, the recombinant plasmid was proved to be true. The target fragment was obtained by digestion of the plasmid pBS- KS using restrictive enzyme NcoI/BamHI and was inserted into the prokaryotic expression vector pYA3334 containing asd gene and was introduceded into the delta Cya ,delta Crp ,delta Asd attenuated Salmonella typhimurium strain X4550 by electroporation transformations , which is a balanced lethal recombinant. Analyzed the products of expression through SDS-PAGE and Western-blotting . The Immune Effect of Recombinant strain X4550(pYA3334-K88ac-STII) was indicated by the level of antibody and pathological change. Mice were then inoculated with 109 or 1010 cfu recombination strain through a stainless steel gavage tube,as experimentation vaccine, empty vector X4550(pYA3334)and PBS were used as control.Mice of three groups were immunized with each of them and challenged with virulent strain C83915(STⅡ﹢).
Result: The fusion gene was coloned into prokaryotic expression vector pYA3334 with the right ORF, and can express the products expected successfully in X4550. The recombinant strain can express fusion protein, which has the same molecular weight as prospective protein.The fusion protein expressed by recombinant strain X4550(pYA3334-K88ac-STII) can be identified by anti-K88ac.No corresponding protein was expressed in X4550 transformed by pYA3334 .The stability of The recombinant strain X4550(pYA3334-K88ac-STII) in vitro asserted The foreign antigen K88ac-STⅡcan be expressed stabily. The mice immunized with recombinant strain X4550(pYA3334-K88ac-STII)vaccine can produce anti- K88ac-STⅡ.Recombinant strain X4550(pYA3334-K88ac-STII) has effective Immune Effect to mice. The recombinant strain is worth considering as a candidate vaccine strain against enterotoxin Escherichia coli disease and Salmonella typhimurium infections.
根据GenBank中登录号为m29375的K88ac基因序列以及减毒鼠伤寒沙门氏菌原核表达载体pYA3334的多克隆位点设计特异性引物P1、P2,并在上下游引物的5’端分别引入NcoI和BamHI酶切位点,从大肠杆菌DE3(pET- K88ac-STⅡ)中通过聚合酶链式反应(PCR)扩增得到K88ac-STⅡ融合基因。回收纯化后将K88ac-STⅡ融合基因用T4DNA连接酶连于克隆载体pBS-T上,热击转化受体大肠杆菌Top10,蓝白斑筛选阳性菌落,酶切鉴定证明其中含有K88ac-STⅡ融合基因片段,再测序鉴定该融合基因序列的正确性,命名为pBS-K88ac-STⅡ。之后采用缺失腺苷酸环化酶基因(Δcya)、环腺苷酸受体蛋白基因(Δcrp)以及天冬氨酸β-半醛脱氢酶基因(Δasd)的鼠伤寒沙门氏菌(X4550)作为宿主,将编码K88ac-STII的融合基因切下插入Asd+组成型表达载体pYA3334中,鉴定后通过电转化引入宿主菌X4550中,再次酶切鉴定正确后,过夜培养进行SDS-PAGE和Western-Blot试验,检测其特异性蛋白表达情况。最后分别以重组菌株X4550(pYA3334-K88ac-STII)灌服小鼠作为试验组,以转化了空载体的菌株X4550(pYA3334)作为空载体对照组,同时设PBS空白对照组,分别对三个分组的小鼠进行免疫,采集血清检测其抗体效价,之后免疫小鼠用强毒株C83915(STⅡ﹢)攻击,观察结果。
本实验成功构建了表达K88ac-STII融合基因平衡致死的减毒鼠伤寒沙门氏重组菌株X4550(pYA3334-K88ac-STII),该重组菌株在33Kda处有特异性表达的蛋白条带,与预期分子量大小一致,而空载体对照菌X4550(pYA3334)在该处未有相应条带出现。Western-blot免疫印迹结果显示重组菌株X4550(pYA3334-K88ac-STII)表达的蛋白能够被K88ac阳性血清特异性结合,在相应位置出现特异性显色条带。这证明重组菌株能够表达K88ac-STII特异性融合蛋白,重组菌的体外稳定实验也证明其有良好的稳定性,从而保证了K88ac-STII抗原能够得以持续表达。而且试验小鼠灌喂重组减毒鼠伤寒沙门氏菌X4550 (pYA3334-K88ac-STII)后,能够产生特异性的抗体,而且对大肠杆菌强毒株具有一定的抵抗力。这为进一步研制相应的抗仔猪大肠杆菌病及相应的沙门氏菌病的双价活疫苗侯选株奠定了重要基础。
Object:To construct a live oral Salmonella typhimurium vaccine strain expressing K88ac-STII fusion gene of enterotoxigenic Escherichia coli.
Methods: Primers(P1,P2) were designed according to the nucleotide sequence of K88ac gene of enterotoxigenic Escherichia coli(ETEC) reported respectively and the expression vector pYA3334 . With the specific primers, the gene encoding the fusion protein K88ac-STII was amplified by PCR of the plasmid pET- K88ac-STⅡand was inserted into the linearized plasmid vector PBS-T. By enzyme analysis, PCR and sequencing, the recombinant plasmid was proved to be true. The target fragment was obtained by digestion of the plasmid pBS- KS using restrictive enzyme NcoI/BamHI and was inserted into the prokaryotic expression vector pYA3334 containing asd gene and was introduceded into the delta Cya ,delta Crp ,delta Asd attenuated Salmonella typhimurium strain X4550 by electroporation transformations , which is a balanced lethal recombinant. Analyzed the products of expression through SDS-PAGE and Western-blotting . The Immune Effect of Recombinant strain X4550(pYA3334-K88ac-STII) was indicated by the level of antibody and pathological change. Mice were then inoculated with 109 or 1010 cfu recombination strain through a stainless steel gavage tube,as experimentation vaccine, empty vector X4550(pYA3334)and PBS were used as control.Mice of three groups were immunized with each of them and challenged with virulent strain C83915(STⅡ﹢).
Result: The fusion gene was coloned into prokaryotic expression vector pYA3334 with the right ORF, and can express the products expected successfully in X4550. The recombinant strain can express fusion protein, which has the same molecular weight as prospective protein.The fusion protein expressed by recombinant strain X4550(pYA3334-K88ac-STII) can be identified by anti-K88ac.No corresponding protein was expressed in X4550 transformed by pYA3334 .The stability of The recombinant strain X4550(pYA3334-K88ac-STII) in vitro asserted The foreign antigen K88ac-STⅡcan be expressed stabily. The mice immunized with recombinant strain X4550(pYA3334-K88ac-STII)vaccine can produce anti- K88ac-STⅡ.Recombinant strain X4550(pYA3334-K88ac-STII) has effective Immune Effect to mice. The recombinant strain is worth considering as a candidate vaccine strain against enterotoxin Escherichia coli disease and Salmonella typhimurium infections.
引文
[1] 李决.兽医微生物学及免疫学[M],第 1 版.成都:四川科学技术出版社,1994.6.
[2] 蔡宝祥.家畜传染病学[M].第 4 版.北京:中国农业出版社,2001.7.
[3] 伍建宏,叶夫芸,徐建国.致泻性大肠杆菌中发现小肠结肠炎郁尔森菌毒力岛[J].中华微生物学和免疫学杂志,2000,20(5).
[4] 应天翼,韩照中,冯尔玲等.肠毒素性大肠杆菌 CS6 菌毛抗原与霍乱毒素 B 亚基在痢疾菌中的表达及其免疫学效果[J].生物工程学报,2001,17(1):29-33.
[5] 董国雄,张英,江美娟.对 FC 株猪源性肠毒素大肠杆菌致病因子的研究[J].江苏农学院学报,1986,7(3):1-6
[6] 李毅,抗大肠埃希氏菌 K88 黏附素单克隆抗体在仔猪黄痢治疗中的初步应用[J].中国畜禽传染病,1989,(5):34-35
[7] 刘书亮,王红宁,陶勇等,四川省规模化猪场仔猪黄白痢 E.coli 分离鉴定[J].中国预防兽医学报,2001,23(5):267-371
[8] 王红琳,熊忠良,周绍风等.湖北省仔猪致病性大肠杆菌血清型鉴定[J].动物医学进展,2002,23(3):68-69
[9] 姜卫东,黄引贤,广东省仔猪腹泻大肠杆菌血清型鉴定[J].中国兽医学报,1998,18(1):64-66
[10] 袁万哲,何孔旺,陆承平等.产肠毒素性大肠杆菌主要毒力因子的研究进展[J]动物医学进展,2005,26(2):6-9.
[11] 王远志,产肠毒素性大肠杆菌融合基因的克隆与原核表达[D].新疆:新疆农业大学,2004,6.
[12] 张雪寒,何孔旺,张书霞.产肠毒素性大肠杆菌肠毒素的研究概况[J].动物医学进展.2003, 24 (3),38-40.
[13] 宋秉生,王兴亚.仔猪人肠杆凶性腹泻[J].甘肃畜牧兽医,1997,3:22-26
[14] 李文建,邹全明.黏膜免疫佐剂:肠产肠毒素性大肠杆菌不耐热肠毒素(LT)研究进展[J].免疫学杂志,2000,16(4):85-87.
[15] 毛旭虎,邹全明. 大肠杆菌不耐热肠毒素的分子生物学特征[J].国外医学临床生物化学与检验学分册,2000,21(5):228-229.
[16] 王嘉福,陆承平. 猪大肠杆菌耐热肠毒素和热敏肠毒素基因的融合及表达[J].微生物学报.2003.43(1):253-255.
[17] 徐兵,张兆山,李淑琴,等.产肠毒素大肠杆菌的热敏肠毒素 B 亚基(LT-B)和耐热肠毒素(ST)基因融合及其表达[J] 生物工程学报,1999,15(4)440-443.
[18] 许崇波,冯书章,冯书章,等.大肠杆菌耐热性肠毒素 I 基因核苷酸序列分析与免疫原性研究[J] 中国兽医学报,1996,16(4):327.
[19] 郭志儒(摘引).第一个获准上市的疫苗[J].中国兽医学报.2005.25(5):469.
[20] 陈瑛,贾桂珍,陈新萍.新型动物免疫疫苗研究概况[J].贵州畜牧兽医.2004,28(5):11-13.
[21] 彭健,王劼 ,詹志春等.预防仔猪大肠杆菌性腹泻卵黄抗体的研究进展[J].饲料工业.2005,6.
[22] 朱庆义.致泻大肠埃希菌及其毒力岛研究进展[J].中华检验医学杂志.2003,26(10).
[23] 李毅,刘秀梵.抗大肠埃希氏菌 K88ab,K88ac 和 K88ad 特异单克隆抗体[J].微生物学报,1989,29(5):348-353.
[24] 高崧,刘秀梵.抗大肠埃希氏菌黏附素单克隆抗体诊断试剂的研制及其现场初步应用[J].畜牧兽医学报.1993,24(3):248-253.
[25] Nataro JP, Kaper JB. Diarrheagenic Escherichia coli[J].Clinical Microbiol Review,1998, 11(1):142-201.
[26] Henton MM , Engellbrecht MM , Escherichia coli serotypes in pigs in south Africa[J].Onderstepoort J Vet Res,1997,64(3):175-187
[27] Wei Guang-sen,Xu Chong-bo,Fengshuzhan,et al. Effect of ImmunoPotentiator LI on Immunity to Chicken New Castle Disease[J].Journal of HAFLRU,1994,7(4):96-98.
[28] Xuchong-bo,WeiGuang-sen,Fengshuzhan,et al.Construction of FusionGene for Entero -toxigenic Escherichia coli heat-stable Enterotoxin I and Its immunication [J], Acta veterinariaet zootechnica,1997,28(4):330-335.
[29] XuChong-bo,WeiGuang-sen,WangZhuo,et al.Cloning and Nucleotide Sequencing of the Heat-labile Enterotoxin B Subunit Gene in Escherichia Coli[J].Animal Husbandry & Veterinary Medcine,1997,29(4):154-156.
[30] Chauhan D,Pandey P,Ogata A,et al, Cytochrome c-dependent and -independent induction of apoptosis in multiple myeloma cells. J Biol Chem 1997,272:29995-29997
[31] XuChong-bo,WeiGuang-sen,WangZhuo,et al.High-Level ExPression of Fusion Gene of the Heat-stable Enterotoxin(STI)and Heat-labile Enterotoxin B Subunit(LT)of Escherichia Coli[J].Journal of hygiene research, 1998, 27: 13-16.
[32] RoskovI,RoskovF,Serology of Escherichia coli fimbriae[J].Prog Allergy,1983,33:80-105.
[33] Broeck W V, CoxE, Cudega B, etal.The F4 fimbrial antigen of Esherichia coli and its receptors[J].Vet Microb,2000,71:223-244.
[34] Guinee PAM,Jansen W H.Behavior of Escherichia colik antigens K88ab,K88ac,and K88ad inimmuno-electrophoresis,double diffusion, and hemagglutination[J].Infect Immun , 1979,23:700-705.
[35] Lee JH,ISaacson R E.ExPressinn of the gene cluster associated with the Escherichia Coli adhesin K99[J],1995,63(10),4143-4149.
[36] Lo-Tseng T,Lee J, Issacson R E. Regulators of Escherichina coli K99 regionl genes[J].Adv Exp Med Biol,1997,412:303-301.
[37] Grange P A,Mouricout M.Susceptibility of infant mice to F5( K99) E.coli infection: differences in glycosylt ransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains[J] . Glycoconj J,1996,13(1):45-52.
[38] Mol O,Fokkema H,Oudega B.The Escherichia coli K99 periplasmic chaperone FanE is a monomeric protein[J] . FEMS Microbiol Lett ,1996,138(223):185-189.
[39] Jiangcheng Cao,A.Slalam Khan,Manfred E. Ordered Translocation of 987P Fimbrial Subunits through the outer Membrane of Escherichia coli[J].Journal of Bacteriolgy,1995,3704-3713.
[40] Takeda T, Takeda Y,Miwatani T, Genetic labeling of an Ent plasmid that encodes heat-stable enterotoxin of enterotoxigenic Escherichia coli isolated from patients[J].Biken J, 1981,24(4):127-135.
[41] Smith H,Interpretation of results obtained by activation analysis[J].Jforensic SciSoc,1969,9(3):205-209.
[42] Evans DG, Evans DJ Jr,Pierce NF,Differences in the response of rabbit small intesrine to heat-labile and heat-stable enterotoxins of Escherichia coli [J].Infect Immun, 1973,7(6): 873-880.
[43] Edwards R A, Schifferli D M.Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues[J].mol Microbiol,1997,25(4):797-809.
[44] Guzman-Vordazco L M, Export and processing analysis of a fusion between the extrocellular heat-stable enterotoxin ang the periplasmic B-subunite of the heat-labile enterotoxin in E.coil[J].MOL Microbiol,1990,4(2):253-264.
[45] Guzman-verduzco L M, Kupwearoch Y M. Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit[J].Bacteriol ,1987,169:52-58.
[46] Glement J D. Construction of a nontoxin fusion peptide for immunication against Escherichia coli strain that produce heat-labile and heat-stable enterotoxins[J]. Immun, 1990,58:1159
[1] 钱峰 以减毒沙门氏菌为载体的疫苗研究[J].国外医学寄生病分册,1998,25(1):9-13.
[2] 武军元,许程剑,陈创夫 核酸疫苗的运送载体一减毒鼠伤寒沙门氏菌[J].动物医学进展.2OO5,26(11):105—107.
[3] 梁雪芽, 宋厚辉, 江玲丽等 沙门氏菌的基因工程减毒以及在 DNA 疫苗载体中的应用[J].中国兽医杂志.2002,38(7):35-37.
[4] 张辉,焦新安,潘志明,张晓明 减毒鼠伤寒沙门氏菌运送 CD8 + T 细胞表位的细胞免疫应答[J].细胞与分子免疫学杂志 ,2006,(02):137-140.
[5]张晓明,潘志明 运送 DNA 疫苗的减毒沙门氏菌载体系统[J].微生物学免疫学进展.2002, 30 (4):54-60.
[6] 周宗安.王延茹.基因疫苗的研究进展及临床应用[J].东南国防医药.2003.5(2):99-102.
[7] 王剑虹.沙门菌减毒活菌苗国外医学[J].预防.诊断.治疗用生物制品分册.2000.23(5):197-199.
[8] 李决.兽医微生物学及免疫学[M],第 1 版.成都:四川科学技术出版社,1994.6.
[9] 李文桂,陈雅棠 细菌重组减毒鼠伤寒沙门氏菌疫苗的研究进展[J].动物医学进展.2004,25(2):49-53.
[10] 马有智,戴贤君,李肖梁等.构建成了携带表达猪链球菌溶血素基因的减毒沙门氏菌的构建及鉴定[J].中国兽医学报.2005.25(5):478-480.
[11] 应天翼,韩照中,冯尔玲等.肠毒素性大肠杆菌CS6菌毛抗原与霍乱毒素B亚基在痢疾菌中的表达及其免疫学效果[J].生物工程学报,2001,17(1):29-33.
[12] 彭健,王劼,詹志春等 预防仔猪大肠杆菌性腹泻卵黄抗体的研究进展[J].饲料工业.2005,6.
[13] 谢明权,覃宗华,蔡建平等 EnMIC2 重组减毒沙门氏菌的构建及免疫保护效果研究[J].畜牧兽医学报.2005,36(7):705-710.
[14] 王芳,焦新安,潘志明等 融合表达淋巴细胞脉络丛脑膜炎病毒和卵清白蛋白CD8+T细胞表位的减毒鼠伤寒沙门氏菌的构建与鉴定[J].中国预防兽医学报.2004,26(1):14-17.
[15] Wolff J A , Malone R W, Williams P , etal . Direct gene transfer in to mouse muscle in vivo[J]. Scinece , 1990 , 247 : 1465–1468.
[16] Mary Jo Wick. The role of dendritic cells in the immune response to Salmonella[J]. Immunology Letters.2003, 85 : 99-102.
[17] Dietrich G, Spreng S , Gentschev I , etal. Bacterial systems for thedeliveryof eukaryotic antigen expression vectors[J]. Antisense NucleicAcid Drug Dev,2000, 10(5):391-399.
[18] Ikonomidis G, Portnoy DA, Gerhardwetal. Influenza-specific immunity induced by recombinantListeria monocytogenes vaccines[J].Vaccine . 1997,15(4): 433-440.
[19] Stover CK, dela Cruz VF, Fuerst TR, etal. New use of BCG for recombinant vaccines[J]. Nature.1991, 351(6326): 456-460.
[20] Kremer L, Dupre L, Riveau G, etal. Systemic and mucosal immune responses after intranasaladministration of recombinant Mycobacterium bovis bacillus Calmette-Guerin expressing glutathioneS-transferase from Schistosoma haematobium [J]. Infect Immun. 1998, 66 (12): 5669-5676.
[21] Noriega FR, Losonsky G, Wang JY, et al. Further characterization of delta aroA delta virG Shigellaflexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for deliveringantigens of enterotoxigenic Escherichia coli[J]. Infect Immun. 1996, 64(1): 23-27.
[22] Mary Jo Wick. The role of dendritic cells in the immune response to Salmonella[J]. Immunology Letters.2003, 85 : 99-102.
[23] Aifang Du*, Suhua Wang. Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens[J]. International Journal for Parasitology ,2005,35:777–785.
[24] Cardenas Letal[J].Clin Microbiol Rev.1992,5(3):328-342.
[25] Curtiss R, Hassan J1 Nonrecombinant and recombinantavirulent Salmonella vaccines for poultry[J].Vet ImmunolImmunopathol, 1996, 54 (124) : 365-372.
[26] Gunn J S, Ryan S S, VanVelk inburgh J C, etal1 Genetic andfunctional analysis of a PmrA–PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium [J]. Infect Immun, 2000, 68 (11) : 6139-6146.
[27] Douglas M , Robert L , David A , etal.Dietary formulation with rendered spent henmeals on a total amino acid versus a digestible amino acid basis [J]. Poult Sci, 1999, 284: 967-970.
[28] Heithoff D M , Sinsheimer R L , Low D A , etal. A n essential role for DNA adenine methylation in bacterial virulence [J].Trans R Soc Lond B Biol Sci, 2000, 355 (1397) : 633-642.
[29] Sirard JC, Niedergang F, Kraehenbuhl JP. Live atenuated Salmonella: aparadigm of mucosal vaccines[J]. Immunol Rev. 1999, 171:5-26
[30]Rescigno M, Rotta G, Valzasina B, etal. Dendritic cells shuttle microbes across gut epithelial monolayers[J]. Immunobiology. 2001, 204(5): 572-581.
[31] Hopkins SA, Niedergang F, Corthesy-Tbeulaz IF, etal. A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells[J]. Cell Microbial. 2000,2(1): 59-68。
[32] Victòria E Sevil Domènech , Klaus Panthel , Katrin M. Meinel,etal. Rapid clearance of a recombinant Salmonella vaccine carrier preventsenhanced antigen -specific CD8 T-cell responses after oral boost immunizations [J].Microbes and Infection, 2005,7:860–866.
[33] Kaoru Geddes, Micah Worley, George Niemann,etal.etal Identification of New Secreted Effectors in Salmonella enterica Serovar Typhimurium[J] Infection and Immunity,2005, 73: 6260–6271.
[34] Ruan L,Wang YQiang BQ.Status and Prosoect for New Gneration Vaccine Development[J].Beijing:Science press,1992:171-183.
[35] Heithoff D M , Sinsheimer R L , Low D A , etal. A n essential role for DNA adenine methylation in bacterial virulence [J].Trans R Soc Lond B Biol Sci, 2000, 355 (1397) : 633-642.
[36] Douglas M , Robert L , David A , etal.Dietary formulation with rendered spent henmeals on a total amino acid versus a digestible amino acid basis [J]. Poult Sci, 1999, 284: 967-970.
[1] 应天翼,韩照中,冯尔玲等.肠毒素性大肠杆菌 CS6 菌毛抗原与霍乱毒素 B 亚基在痢疾菌中的表达及其免疫学效果[J].生物工程学报,2001,17(1):29-33.
[2] 袁万哲,何孔旺,陆承平等.产肠毒素性大肠杆菌主要毒力因子的研究进展[J]动物医学进展,2005,26(2):6-9.
[3] RoskovI,RoskovF,Serology of Escherichia coli fimbriae[J].Prog Allergy,1983, 33:80-105.
[4] Guinee PAM,Jansen W H.Behavior of Escherichia colik antigens K88ab,K88ac,and K88ad inimmuno-electrophoresis,double diffusion, and hemagglutination[J]. Infect Immun, 1979,23:700-705.
[5] J . 萨姆布鲁克, D.W. 拉塞尔著. 黄培堂,王嘉玺等译.分子克隆实验指南[M]. 第三版. 北京:科学出版社, 2002. 889.
[6] 姜卫东,黄引贤.广东省仔猪腹泻大肠杆菌血清型鉴定[J].中国兽医学报,1998,18(l):64-66.
[7] 蔡葵蒸,靳国琴,柴君秀,等.仔猪大肠杆菌性腹泻病的调查及病原特性的研[J].动物医学进展.2002,23(5):101-103.
[8] Shin S J,Chang Y F,TimourM,et al.Hybridization of clinical Escherichia coli isolates from calves and piglets in New York State with gene probes for enterotoxins(STa, STb,LT) , heal-stable enterotoxin(ST) and adhesion factors(K88,K99,F41,987P)[J]. Vet Microbiol,1994,38(3):217-225.
[9] Garabal J I, Vazguez F, Blanco J,et al. colonization antigens of Escherichia coil strains isolated from piglets in Spani[J]. Vet Microbiol 1997,54(3-4):321-328.
[10] 陈焕勇.大肠杆菌耐热肠毒索的主要特性和分子生物学研究[J].国外医学一微生物学分册,1990,13(4):166-168。
[11] 王远志,产肠毒素性大肠杆菌融合基因的克隆与原核表达[D].新疆:新疆农业大学,2004,6.
[1] 袁万哲,何孔旺,陆承平等.产肠毒素性大肠杆菌主要毒力因子的研究进展[J]动物医学进展,2005,26(2):6-9.
[2] 黄培堂,W.K.Maas .表达具有热稳定肠毒素Ⅱ免疫原性融合蛋白菌株的构建[J].生物工程学报,1988,4(4):263-270
[3] 葛艳,尤永进,徐泉兴等.产肠毒素大肠杆菌肠毒素LTB,STⅠ和STⅡ融合基因的构建与表达研究[J].中国预防兽医学报,2002,24(5):346-348
[4] 王远志,产肠毒素性大肠杆菌融合基因的克隆与原核表达[D].新疆:新疆农业大学,2004,6.
[2] 蔡宝祥.家畜传染病学[M].第 4 版.北京:中国农业出版社,2001.7.
[3] 伍建宏,叶夫芸,徐建国.致泻性大肠杆菌中发现小肠结肠炎郁尔森菌毒力岛[J].中华微生物学和免疫学杂志,2000,20(5).
[4] 应天翼,韩照中,冯尔玲等.肠毒素性大肠杆菌 CS6 菌毛抗原与霍乱毒素 B 亚基在痢疾菌中的表达及其免疫学效果[J].生物工程学报,2001,17(1):29-33.
[5] 董国雄,张英,江美娟.对 FC 株猪源性肠毒素大肠杆菌致病因子的研究[J].江苏农学院学报,1986,7(3):1-6
[6] 李毅,抗大肠埃希氏菌 K88 黏附素单克隆抗体在仔猪黄痢治疗中的初步应用[J].中国畜禽传染病,1989,(5):34-35
[7] 刘书亮,王红宁,陶勇等,四川省规模化猪场仔猪黄白痢 E.coli 分离鉴定[J].中国预防兽医学报,2001,23(5):267-371
[8] 王红琳,熊忠良,周绍风等.湖北省仔猪致病性大肠杆菌血清型鉴定[J].动物医学进展,2002,23(3):68-69
[9] 姜卫东,黄引贤,广东省仔猪腹泻大肠杆菌血清型鉴定[J].中国兽医学报,1998,18(1):64-66
[10] 袁万哲,何孔旺,陆承平等.产肠毒素性大肠杆菌主要毒力因子的研究进展[J]动物医学进展,2005,26(2):6-9.
[11] 王远志,产肠毒素性大肠杆菌融合基因的克隆与原核表达[D].新疆:新疆农业大学,2004,6.
[12] 张雪寒,何孔旺,张书霞.产肠毒素性大肠杆菌肠毒素的研究概况[J].动物医学进展.2003, 24 (3),38-40.
[13] 宋秉生,王兴亚.仔猪人肠杆凶性腹泻[J].甘肃畜牧兽医,1997,3:22-26
[14] 李文建,邹全明.黏膜免疫佐剂:肠产肠毒素性大肠杆菌不耐热肠毒素(LT)研究进展[J].免疫学杂志,2000,16(4):85-87.
[15] 毛旭虎,邹全明. 大肠杆菌不耐热肠毒素的分子生物学特征[J].国外医学临床生物化学与检验学分册,2000,21(5):228-229.
[16] 王嘉福,陆承平. 猪大肠杆菌耐热肠毒素和热敏肠毒素基因的融合及表达[J].微生物学报.2003.43(1):253-255.
[17] 徐兵,张兆山,李淑琴,等.产肠毒素大肠杆菌的热敏肠毒素 B 亚基(LT-B)和耐热肠毒素(ST)基因融合及其表达[J] 生物工程学报,1999,15(4)440-443.
[18] 许崇波,冯书章,冯书章,等.大肠杆菌耐热性肠毒素 I 基因核苷酸序列分析与免疫原性研究[J] 中国兽医学报,1996,16(4):327.
[19] 郭志儒(摘引).第一个获准上市的疫苗[J].中国兽医学报.2005.25(5):469.
[20] 陈瑛,贾桂珍,陈新萍.新型动物免疫疫苗研究概况[J].贵州畜牧兽医.2004,28(5):11-13.
[21] 彭健,王劼 ,詹志春等.预防仔猪大肠杆菌性腹泻卵黄抗体的研究进展[J].饲料工业.2005,6.
[22] 朱庆义.致泻大肠埃希菌及其毒力岛研究进展[J].中华检验医学杂志.2003,26(10).
[23] 李毅,刘秀梵.抗大肠埃希氏菌 K88ab,K88ac 和 K88ad 特异单克隆抗体[J].微生物学报,1989,29(5):348-353.
[24] 高崧,刘秀梵.抗大肠埃希氏菌黏附素单克隆抗体诊断试剂的研制及其现场初步应用[J].畜牧兽医学报.1993,24(3):248-253.
[25] Nataro JP, Kaper JB. Diarrheagenic Escherichia coli[J].Clinical Microbiol Review,1998, 11(1):142-201.
[26] Henton MM , Engellbrecht MM , Escherichia coli serotypes in pigs in south Africa[J].Onderstepoort J Vet Res,1997,64(3):175-187
[27] Wei Guang-sen,Xu Chong-bo,Fengshuzhan,et al. Effect of ImmunoPotentiator LI on Immunity to Chicken New Castle Disease[J].Journal of HAFLRU,1994,7(4):96-98.
[28] Xuchong-bo,WeiGuang-sen,Fengshuzhan,et al.Construction of FusionGene for Entero -toxigenic Escherichia coli heat-stable Enterotoxin I and Its immunication [J], Acta veterinariaet zootechnica,1997,28(4):330-335.
[29] XuChong-bo,WeiGuang-sen,WangZhuo,et al.Cloning and Nucleotide Sequencing of the Heat-labile Enterotoxin B Subunit Gene in Escherichia Coli[J].Animal Husbandry & Veterinary Medcine,1997,29(4):154-156.
[30] Chauhan D,Pandey P,Ogata A,et al, Cytochrome c-dependent and -independent induction of apoptosis in multiple myeloma cells. J Biol Chem 1997,272:29995-29997
[31] XuChong-bo,WeiGuang-sen,WangZhuo,et al.High-Level ExPression of Fusion Gene of the Heat-stable Enterotoxin(STI)and Heat-labile Enterotoxin B Subunit(LT)of Escherichia Coli[J].Journal of hygiene research, 1998, 27: 13-16.
[32] RoskovI,RoskovF,Serology of Escherichia coli fimbriae[J].Prog Allergy,1983,33:80-105.
[33] Broeck W V, CoxE, Cudega B, etal.The F4 fimbrial antigen of Esherichia coli and its receptors[J].Vet Microb,2000,71:223-244.
[34] Guinee PAM,Jansen W H.Behavior of Escherichia colik antigens K88ab,K88ac,and K88ad inimmuno-electrophoresis,double diffusion, and hemagglutination[J].Infect Immun , 1979,23:700-705.
[35] Lee JH,ISaacson R E.ExPressinn of the gene cluster associated with the Escherichia Coli adhesin K99[J],1995,63(10),4143-4149.
[36] Lo-Tseng T,Lee J, Issacson R E. Regulators of Escherichina coli K99 regionl genes[J].Adv Exp Med Biol,1997,412:303-301.
[37] Grange P A,Mouricout M.Susceptibility of infant mice to F5( K99) E.coli infection: differences in glycosylt ransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains[J] . Glycoconj J,1996,13(1):45-52.
[38] Mol O,Fokkema H,Oudega B.The Escherichia coli K99 periplasmic chaperone FanE is a monomeric protein[J] . FEMS Microbiol Lett ,1996,138(223):185-189.
[39] Jiangcheng Cao,A.Slalam Khan,Manfred E. Ordered Translocation of 987P Fimbrial Subunits through the outer Membrane of Escherichia coli[J].Journal of Bacteriolgy,1995,3704-3713.
[40] Takeda T, Takeda Y,Miwatani T, Genetic labeling of an Ent plasmid that encodes heat-stable enterotoxin of enterotoxigenic Escherichia coli isolated from patients[J].Biken J, 1981,24(4):127-135.
[41] Smith H,Interpretation of results obtained by activation analysis[J].Jforensic SciSoc,1969,9(3):205-209.
[42] Evans DG, Evans DJ Jr,Pierce NF,Differences in the response of rabbit small intesrine to heat-labile and heat-stable enterotoxins of Escherichia coli [J].Infect Immun, 1973,7(6): 873-880.
[43] Edwards R A, Schifferli D M.Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues[J].mol Microbiol,1997,25(4):797-809.
[44] Guzman-Vordazco L M, Export and processing analysis of a fusion between the extrocellular heat-stable enterotoxin ang the periplasmic B-subunite of the heat-labile enterotoxin in E.coil[J].MOL Microbiol,1990,4(2):253-264.
[45] Guzman-verduzco L M, Kupwearoch Y M. Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit[J].Bacteriol ,1987,169:52-58.
[46] Glement J D. Construction of a nontoxin fusion peptide for immunication against Escherichia coli strain that produce heat-labile and heat-stable enterotoxins[J]. Immun, 1990,58:1159
[1] 钱峰 以减毒沙门氏菌为载体的疫苗研究[J].国外医学寄生病分册,1998,25(1):9-13.
[2] 武军元,许程剑,陈创夫 核酸疫苗的运送载体一减毒鼠伤寒沙门氏菌[J].动物医学进展.2OO5,26(11):105—107.
[3] 梁雪芽, 宋厚辉, 江玲丽等 沙门氏菌的基因工程减毒以及在 DNA 疫苗载体中的应用[J].中国兽医杂志.2002,38(7):35-37.
[4] 张辉,焦新安,潘志明,张晓明 减毒鼠伤寒沙门氏菌运送 CD8 + T 细胞表位的细胞免疫应答[J].细胞与分子免疫学杂志 ,2006,(02):137-140.
[5]张晓明,潘志明 运送 DNA 疫苗的减毒沙门氏菌载体系统[J].微生物学免疫学进展.2002, 30 (4):54-60.
[6] 周宗安.王延茹.基因疫苗的研究进展及临床应用[J].东南国防医药.2003.5(2):99-102.
[7] 王剑虹.沙门菌减毒活菌苗国外医学[J].预防.诊断.治疗用生物制品分册.2000.23(5):197-199.
[8] 李决.兽医微生物学及免疫学[M],第 1 版.成都:四川科学技术出版社,1994.6.
[9] 李文桂,陈雅棠 细菌重组减毒鼠伤寒沙门氏菌疫苗的研究进展[J].动物医学进展.2004,25(2):49-53.
[10] 马有智,戴贤君,李肖梁等.构建成了携带表达猪链球菌溶血素基因的减毒沙门氏菌的构建及鉴定[J].中国兽医学报.2005.25(5):478-480.
[11] 应天翼,韩照中,冯尔玲等.肠毒素性大肠杆菌CS6菌毛抗原与霍乱毒素B亚基在痢疾菌中的表达及其免疫学效果[J].生物工程学报,2001,17(1):29-33.
[12] 彭健,王劼,詹志春等 预防仔猪大肠杆菌性腹泻卵黄抗体的研究进展[J].饲料工业.2005,6.
[13] 谢明权,覃宗华,蔡建平等 EnMIC2 重组减毒沙门氏菌的构建及免疫保护效果研究[J].畜牧兽医学报.2005,36(7):705-710.
[14] 王芳,焦新安,潘志明等 融合表达淋巴细胞脉络丛脑膜炎病毒和卵清白蛋白CD8+T细胞表位的减毒鼠伤寒沙门氏菌的构建与鉴定[J].中国预防兽医学报.2004,26(1):14-17.
[15] Wolff J A , Malone R W, Williams P , etal . Direct gene transfer in to mouse muscle in vivo[J]. Scinece , 1990 , 247 : 1465–1468.
[16] Mary Jo Wick. The role of dendritic cells in the immune response to Salmonella[J]. Immunology Letters.2003, 85 : 99-102.
[17] Dietrich G, Spreng S , Gentschev I , etal. Bacterial systems for thedeliveryof eukaryotic antigen expression vectors[J]. Antisense NucleicAcid Drug Dev,2000, 10(5):391-399.
[18] Ikonomidis G, Portnoy DA, Gerhardwetal. Influenza-specific immunity induced by recombinantListeria monocytogenes vaccines[J].Vaccine . 1997,15(4): 433-440.
[19] Stover CK, dela Cruz VF, Fuerst TR, etal. New use of BCG for recombinant vaccines[J]. Nature.1991, 351(6326): 456-460.
[20] Kremer L, Dupre L, Riveau G, etal. Systemic and mucosal immune responses after intranasaladministration of recombinant Mycobacterium bovis bacillus Calmette-Guerin expressing glutathioneS-transferase from Schistosoma haematobium [J]. Infect Immun. 1998, 66 (12): 5669-5676.
[21] Noriega FR, Losonsky G, Wang JY, et al. Further characterization of delta aroA delta virG Shigellaflexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for deliveringantigens of enterotoxigenic Escherichia coli[J]. Infect Immun. 1996, 64(1): 23-27.
[22] Mary Jo Wick. The role of dendritic cells in the immune response to Salmonella[J]. Immunology Letters.2003, 85 : 99-102.
[23] Aifang Du*, Suhua Wang. Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens[J]. International Journal for Parasitology ,2005,35:777–785.
[24] Cardenas Letal[J].Clin Microbiol Rev.1992,5(3):328-342.
[25] Curtiss R, Hassan J1 Nonrecombinant and recombinantavirulent Salmonella vaccines for poultry[J].Vet ImmunolImmunopathol, 1996, 54 (124) : 365-372.
[26] Gunn J S, Ryan S S, VanVelk inburgh J C, etal1 Genetic andfunctional analysis of a PmrA–PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium [J]. Infect Immun, 2000, 68 (11) : 6139-6146.
[27] Douglas M , Robert L , David A , etal.Dietary formulation with rendered spent henmeals on a total amino acid versus a digestible amino acid basis [J]. Poult Sci, 1999, 284: 967-970.
[28] Heithoff D M , Sinsheimer R L , Low D A , etal. A n essential role for DNA adenine methylation in bacterial virulence [J].Trans R Soc Lond B Biol Sci, 2000, 355 (1397) : 633-642.
[29] Sirard JC, Niedergang F, Kraehenbuhl JP. Live atenuated Salmonella: aparadigm of mucosal vaccines[J]. Immunol Rev. 1999, 171:5-26
[30]Rescigno M, Rotta G, Valzasina B, etal. Dendritic cells shuttle microbes across gut epithelial monolayers[J]. Immunobiology. 2001, 204(5): 572-581.
[31] Hopkins SA, Niedergang F, Corthesy-Tbeulaz IF, etal. A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells[J]. Cell Microbial. 2000,2(1): 59-68。
[32] Victòria E Sevil Domènech , Klaus Panthel , Katrin M. Meinel,etal. Rapid clearance of a recombinant Salmonella vaccine carrier preventsenhanced antigen -specific CD8 T-cell responses after oral boost immunizations [J].Microbes and Infection, 2005,7:860–866.
[33] Kaoru Geddes, Micah Worley, George Niemann,etal.etal Identification of New Secreted Effectors in Salmonella enterica Serovar Typhimurium[J] Infection and Immunity,2005, 73: 6260–6271.
[34] Ruan L,Wang YQiang BQ.Status and Prosoect for New Gneration Vaccine Development[J].Beijing:Science press,1992:171-183.
[35] Heithoff D M , Sinsheimer R L , Low D A , etal. A n essential role for DNA adenine methylation in bacterial virulence [J].Trans R Soc Lond B Biol Sci, 2000, 355 (1397) : 633-642.
[36] Douglas M , Robert L , David A , etal.Dietary formulation with rendered spent henmeals on a total amino acid versus a digestible amino acid basis [J]. Poult Sci, 1999, 284: 967-970.
[1] 应天翼,韩照中,冯尔玲等.肠毒素性大肠杆菌 CS6 菌毛抗原与霍乱毒素 B 亚基在痢疾菌中的表达及其免疫学效果[J].生物工程学报,2001,17(1):29-33.
[2] 袁万哲,何孔旺,陆承平等.产肠毒素性大肠杆菌主要毒力因子的研究进展[J]动物医学进展,2005,26(2):6-9.
[3] RoskovI,RoskovF,Serology of Escherichia coli fimbriae[J].Prog Allergy,1983, 33:80-105.
[4] Guinee PAM,Jansen W H.Behavior of Escherichia colik antigens K88ab,K88ac,and K88ad inimmuno-electrophoresis,double diffusion, and hemagglutination[J]. Infect Immun, 1979,23:700-705.
[5] J . 萨姆布鲁克, D.W. 拉塞尔著. 黄培堂,王嘉玺等译.分子克隆实验指南[M]. 第三版. 北京:科学出版社, 2002. 889.
[6] 姜卫东,黄引贤.广东省仔猪腹泻大肠杆菌血清型鉴定[J].中国兽医学报,1998,18(l):64-66.
[7] 蔡葵蒸,靳国琴,柴君秀,等.仔猪大肠杆菌性腹泻病的调查及病原特性的研[J].动物医学进展.2002,23(5):101-103.
[8] Shin S J,Chang Y F,TimourM,et al.Hybridization of clinical Escherichia coli isolates from calves and piglets in New York State with gene probes for enterotoxins(STa, STb,LT) , heal-stable enterotoxin(ST) and adhesion factors(K88,K99,F41,987P)[J]. Vet Microbiol,1994,38(3):217-225.
[9] Garabal J I, Vazguez F, Blanco J,et al. colonization antigens of Escherichia coil strains isolated from piglets in Spani[J]. Vet Microbiol 1997,54(3-4):321-328.
[10] 陈焕勇.大肠杆菌耐热肠毒索的主要特性和分子生物学研究[J].国外医学一微生物学分册,1990,13(4):166-168。
[11] 王远志,产肠毒素性大肠杆菌融合基因的克隆与原核表达[D].新疆:新疆农业大学,2004,6.
[1] 袁万哲,何孔旺,陆承平等.产肠毒素性大肠杆菌主要毒力因子的研究进展[J]动物医学进展,2005,26(2):6-9.
[2] 黄培堂,W.K.Maas .表达具有热稳定肠毒素Ⅱ免疫原性融合蛋白菌株的构建[J].生物工程学报,1988,4(4):263-270
[3] 葛艳,尤永进,徐泉兴等.产肠毒素大肠杆菌肠毒素LTB,STⅠ和STⅡ融合基因的构建与表达研究[J].中国预防兽医学报,2002,24(5):346-348
[4] 王远志,产肠毒素性大肠杆菌融合基因的克隆与原核表达[D].新疆:新疆农业大学,2004,6.