产肠毒素大肠杆菌多价肠毒素疫苗的研究
|
摘要
产肠毒素大肠杆菌(ETEC)能够引起多种幼龄动物以及婴幼儿的胃肠道感染与严重腹泻,给各国的畜牧养殖业及人类健康带来极大的危害。疫苗接种是预防ETEC感染的有效手段,它避免了抗生素滥用所带来的药物残留及细菌产生耐药性等问题,是多年来重要的研究方向。耐热性肠毒素(ST)是ETEC的重要毒力因子,它是免疫原性很弱的小分子半抗原,如何将ST与大分子载体偶联使其具备良好的免疫原性并消除其天然毒性是ETEC疫苗研究中的难点。现有ETEC疫苗中均不含有ST组分,不能诱导有效的抗毒素免疫,因此免疫保护效果有待提高。
为了获得更有效的ETEC疫苗,本论文的研究目的是将编码两种ST分子(STa、STb)的基因与不耐热肠毒素B亚基(LTB)的基因进行融合,进而制备3价融合ETEC肠毒素的DNA疫苗与蛋白疫苗,其特征是含有3种ETEC重要肠毒素毒力因子(分别为LTB、STa, STb)的抗原表位,能够引起抗3种肠毒素的特异性抗体反应,为机体提供全面的抗毒素免疫,从而提高ETEC疫苗的保护率。本论文主要工作如下：
(1)多价肠毒素DNA疫苗的研究：应用重叠延伸PCR技术扩增获得了鸡胰岛素信号肽的基因ins和鸡尿激酶型血纤维蛋白溶酶原激活剂的信号肽基因upa,将肠毒素基因LTB、STa、STb及信号肽基因进行连接得到多价融合基因,在相邻的肠毒素基因之间设计了编码5-7个氨基酸的连接肽序列,将融合基因插入真核表达载体pCI,获得了3种多价肠毒素真核表达质粒pCI-ins-SLS、pCI-upa-SLS、pCI-SLS。经DNA测序证明,所构建的表达载体目标基因序列正确。利用柱层析法纯化DNA疫苗,经脂质体介导转染体外培养的Hela细胞进行瞬时表达实验,免疫荧光实验的结果表明所构建的真核表达质粒能够表达目的重组蛋白。用3种DNA疫苗免疫产蛋母鸡,ELISA测定表明获得了LTB特异性抗体,但STa及STb特异性抗体的效价较低；用其中不带信号肽的DNA疫苗pCI-SLS免疫小鼠,获得了高水平的抗LTB、STa及STb的特异性血清抗体,该抗血清具有中和STb毒性的活性,但没有STa的中和活性,在小鼠攻毒保护实验中提供了50%的保护率,与对照组相比有显著差别(P<0.05)
(2)多价肠毒素蛋白疫苗的研究：首先利用PCR法扩增获得了三种肠毒素LTB、STa、STb的编码基因,应用“限制性内切酶酶切-连接酶连接”等基因工程手段将肠毒素基因LTB、STa、STb进行连接得到三价融合基因LTB-STa-STb,每两段相邻的基因之间设计了编码7个氨基酸的连接肽序列。又以质粒pCI-SLS为模板扩增得到了三价融合基因STa-LTB-STb。将融合基因克隆入原核表达载体pET-30a,获得了2种三价肠毒素原核表达质粒pET30-LSS和pET30-SLS,转化入宿主菌E. coli BL21(DE3)中后经诱导剂IPTG诱导,聚丙烯酞胺凝胶电泳及蛋白质免疫印迹检测实验表明成功的表达了重组三价肠毒素融合蛋白LSS和SLS,分子量均为21 kDa。使用镍离子琼脂糖凝胶色谱柱进行亲和层析,获得了纯化的重组三价肠毒素蛋白。用两种三价肠毒素蛋白疫苗免疫母鸡及小鼠,通过ELISA法检测抗体效价,结果表明在两种动物都获得了较高水平的抗LTB、STa及STb免疫反应,多价抗毒素能够中和STa及STb的毒性,在小鼠攻毒保护实验中提供了50-70%的保护率,与对照组相比有显著差别(P<0.05)。
综上所述,本论文构建的2种三价肠毒素重组蛋白LSS和SLS,同时具有ETEC的三种肠毒素(LTB、STa及STb)免疫原性,免疫动物可引发全面的保护性抗毒素免疫反应,从而能对所有菌毛类型的ETEC致病菌株产生有效的免疫保护,为广谱性ETEC疫苗的研究打下基础。在本论文构建的三价肠毒素DNA疫苗中,STa及STb的免疫原性有所提高,但低于相应的蛋白抗原。
Enterotoxigenic Escherichia coli (ETEC) strains remain important pathogens causing diarrhea in children and a wide variety of newborn animals. Vaccination is an effective approach to control ETEC-induced infections which avoids the problem of antibiotics residue and antibiotics resistance. Currently available ETEC vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). Heat-stable enterotoxin a (STa) and b (STb) are important virulence fators of ETEC, but they are pooly immunogenic due to their small size. Preparation of ST-carrier toxoids which are immunogenic and devoid of toxicity continues to be a challege for ETEC vaccines development. However, the induction of antitoxic responses against STa and STb has merit, as these two poorly immunogenic toxins are frequently associated with ETEC strains.
In order to obtain more effective ETEC vaccines, the aim of this study was to fuse the three coding genes of enterotoxins LTB, STa, and STb, and construct recombinant trivalent enterotoxin DNA vaccines and protein vaccines, in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. The main work of this thesis was as follows:
(1) The study of polyvalent enterotoxin DNA vaccines:chicken insulin signal peptide gene ins and chicken urokinase type plasminogen activator signal peptide gene upa were amplified by splicing by overlap extension (SOE) PCR. Coding genes for LTB, STa, and STb and signal peptide gene (ins or upa) were fused to yield the polyvalent enterotoxin fusion gene with or without signal peptide gene. Two carefully selected proline-rich natural oligopeptides, PPASP and SASTTPP, were included in the fusion to link the adjacent toxins. Fusion genes were then cloned into eukaryotic expression vector pCI to obtain 3 polyvalent enterotoxin DNA vaccines pCI-ins-SLS、pCI-upa-SLS、pCI-SLS. The DNA sequences of the recombinant plasmids were verified by DNA sequencing. DNA vaccines were purified by column chromatography and used to transfect Hela cells using liposome, then indirect immunofluorescence experiments confirmed the expression of polyvalent enterotoxin in Hela cells by the recombinant plasmids. Immunization of chickens with the 3 DNA vaccines elicited low antibody level to LTB and very weak antibody responses to STa or STb, but the DNA vaccine pCI-SLS elicited a significant elevation in serum antibodies to LTB, STa, and STb in mice and the mice anti-sera were able to neutralize the toxicity of STb but not STa. Mice receiving this DNA vaccine had a survival rate of 50% in the vaccination-challenge study which is significantly higher (P<0.05) than that in the control group.
(2) The study of polyvalent enterotoxin protein vaccines:Coding genes for LTB, STa, and STb were obtained by PCR and fused to yield the polyvalent enterotoxin fusion gene LTB-STa-STb by traditional "restriction enzyme digestion-ligation" method. Two similar 7-amino-acid oligopeptides were included in the fusion to link the adjacent toxins. The other fusion enterotoxin gene of STa-LTB-STb was amplified from plasmid pCI-SLS. These two fusion genes were then cloned into vector pET-30a to produce 2 polyvalent enterotoxin prokaryotic expression plasmids. Fusion enterotoxin proteins, LSS and SLS, with the molecular weight of 21 kDa, were expressed in E. coli BL21 which was confirmed by SDS-PAGE and Western blot using anti-His tag antibodies. Fusion proteins were then purified by nickel affinity chromatography. Immunization of chickens or mice with 2 protein vaccines both elicited a high level of antibody responses to LTB, STa, and STb. The IgY or the mice anti-sera were able to neutralize the toxicity of STa and STb. Mice receiving the protein vaccines had a survival rate of 50-70% in the vaccination-challenge study, which was significantly higher (P<0.05) than that in the control group.
In conclusion, the two recombinant trivalent enterotoxin proteins, LSS and SLS, were immunogenic for all the three enterotoxin components (i.e., LTB, STa, and STb) and were able to induce protective antitoxins in animals. Therefore, they could be included in a multivalent vaccine as a potent component to provide broad-spectrum protection against diarrhea caused by ETEC expressing various fimbriae. Though the immunogenicity of STa and STb was enhanced in the trivalent enterotoxin DNA vaccines constructed in this work, it was still weaker than that in the corresponding recombinant protein.
为了获得更有效的ETEC疫苗,本论文的研究目的是将编码两种ST分子(STa、STb)的基因与不耐热肠毒素B亚基(LTB)的基因进行融合,进而制备3价融合ETEC肠毒素的DNA疫苗与蛋白疫苗,其特征是含有3种ETEC重要肠毒素毒力因子(分别为LTB、STa, STb)的抗原表位,能够引起抗3种肠毒素的特异性抗体反应,为机体提供全面的抗毒素免疫,从而提高ETEC疫苗的保护率。本论文主要工作如下：
(1)多价肠毒素DNA疫苗的研究：应用重叠延伸PCR技术扩增获得了鸡胰岛素信号肽的基因ins和鸡尿激酶型血纤维蛋白溶酶原激活剂的信号肽基因upa,将肠毒素基因LTB、STa、STb及信号肽基因进行连接得到多价融合基因,在相邻的肠毒素基因之间设计了编码5-7个氨基酸的连接肽序列,将融合基因插入真核表达载体pCI,获得了3种多价肠毒素真核表达质粒pCI-ins-SLS、pCI-upa-SLS、pCI-SLS。经DNA测序证明,所构建的表达载体目标基因序列正确。利用柱层析法纯化DNA疫苗,经脂质体介导转染体外培养的Hela细胞进行瞬时表达实验,免疫荧光实验的结果表明所构建的真核表达质粒能够表达目的重组蛋白。用3种DNA疫苗免疫产蛋母鸡,ELISA测定表明获得了LTB特异性抗体,但STa及STb特异性抗体的效价较低；用其中不带信号肽的DNA疫苗pCI-SLS免疫小鼠,获得了高水平的抗LTB、STa及STb的特异性血清抗体,该抗血清具有中和STb毒性的活性,但没有STa的中和活性,在小鼠攻毒保护实验中提供了50%的保护率,与对照组相比有显著差别(P<0.05)
(2)多价肠毒素蛋白疫苗的研究：首先利用PCR法扩增获得了三种肠毒素LTB、STa、STb的编码基因,应用“限制性内切酶酶切-连接酶连接”等基因工程手段将肠毒素基因LTB、STa、STb进行连接得到三价融合基因LTB-STa-STb,每两段相邻的基因之间设计了编码7个氨基酸的连接肽序列。又以质粒pCI-SLS为模板扩增得到了三价融合基因STa-LTB-STb。将融合基因克隆入原核表达载体pET-30a,获得了2种三价肠毒素原核表达质粒pET30-LSS和pET30-SLS,转化入宿主菌E. coli BL21(DE3)中后经诱导剂IPTG诱导,聚丙烯酞胺凝胶电泳及蛋白质免疫印迹检测实验表明成功的表达了重组三价肠毒素融合蛋白LSS和SLS,分子量均为21 kDa。使用镍离子琼脂糖凝胶色谱柱进行亲和层析,获得了纯化的重组三价肠毒素蛋白。用两种三价肠毒素蛋白疫苗免疫母鸡及小鼠,通过ELISA法检测抗体效价,结果表明在两种动物都获得了较高水平的抗LTB、STa及STb免疫反应,多价抗毒素能够中和STa及STb的毒性,在小鼠攻毒保护实验中提供了50-70%的保护率,与对照组相比有显著差别(P<0.05)。
综上所述,本论文构建的2种三价肠毒素重组蛋白LSS和SLS,同时具有ETEC的三种肠毒素(LTB、STa及STb)免疫原性,免疫动物可引发全面的保护性抗毒素免疫反应,从而能对所有菌毛类型的ETEC致病菌株产生有效的免疫保护,为广谱性ETEC疫苗的研究打下基础。在本论文构建的三价肠毒素DNA疫苗中,STa及STb的免疫原性有所提高,但低于相应的蛋白抗原。
Enterotoxigenic Escherichia coli (ETEC) strains remain important pathogens causing diarrhea in children and a wide variety of newborn animals. Vaccination is an effective approach to control ETEC-induced infections which avoids the problem of antibiotics residue and antibiotics resistance. Currently available ETEC vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). Heat-stable enterotoxin a (STa) and b (STb) are important virulence fators of ETEC, but they are pooly immunogenic due to their small size. Preparation of ST-carrier toxoids which are immunogenic and devoid of toxicity continues to be a challege for ETEC vaccines development. However, the induction of antitoxic responses against STa and STb has merit, as these two poorly immunogenic toxins are frequently associated with ETEC strains.
In order to obtain more effective ETEC vaccines, the aim of this study was to fuse the three coding genes of enterotoxins LTB, STa, and STb, and construct recombinant trivalent enterotoxin DNA vaccines and protein vaccines, in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. The main work of this thesis was as follows:
(1) The study of polyvalent enterotoxin DNA vaccines:chicken insulin signal peptide gene ins and chicken urokinase type plasminogen activator signal peptide gene upa were amplified by splicing by overlap extension (SOE) PCR. Coding genes for LTB, STa, and STb and signal peptide gene (ins or upa) were fused to yield the polyvalent enterotoxin fusion gene with or without signal peptide gene. Two carefully selected proline-rich natural oligopeptides, PPASP and SASTTPP, were included in the fusion to link the adjacent toxins. Fusion genes were then cloned into eukaryotic expression vector pCI to obtain 3 polyvalent enterotoxin DNA vaccines pCI-ins-SLS、pCI-upa-SLS、pCI-SLS. The DNA sequences of the recombinant plasmids were verified by DNA sequencing. DNA vaccines were purified by column chromatography and used to transfect Hela cells using liposome, then indirect immunofluorescence experiments confirmed the expression of polyvalent enterotoxin in Hela cells by the recombinant plasmids. Immunization of chickens with the 3 DNA vaccines elicited low antibody level to LTB and very weak antibody responses to STa or STb, but the DNA vaccine pCI-SLS elicited a significant elevation in serum antibodies to LTB, STa, and STb in mice and the mice anti-sera were able to neutralize the toxicity of STb but not STa. Mice receiving this DNA vaccine had a survival rate of 50% in the vaccination-challenge study which is significantly higher (P<0.05) than that in the control group.
(2) The study of polyvalent enterotoxin protein vaccines:Coding genes for LTB, STa, and STb were obtained by PCR and fused to yield the polyvalent enterotoxin fusion gene LTB-STa-STb by traditional "restriction enzyme digestion-ligation" method. Two similar 7-amino-acid oligopeptides were included in the fusion to link the adjacent toxins. The other fusion enterotoxin gene of STa-LTB-STb was amplified from plasmid pCI-SLS. These two fusion genes were then cloned into vector pET-30a to produce 2 polyvalent enterotoxin prokaryotic expression plasmids. Fusion enterotoxin proteins, LSS and SLS, with the molecular weight of 21 kDa, were expressed in E. coli BL21 which was confirmed by SDS-PAGE and Western blot using anti-His tag antibodies. Fusion proteins were then purified by nickel affinity chromatography. Immunization of chickens or mice with 2 protein vaccines both elicited a high level of antibody responses to LTB, STa, and STb. The IgY or the mice anti-sera were able to neutralize the toxicity of STa and STb. Mice receiving the protein vaccines had a survival rate of 50-70% in the vaccination-challenge study, which was significantly higher (P<0.05) than that in the control group.
In conclusion, the two recombinant trivalent enterotoxin proteins, LSS and SLS, were immunogenic for all the three enterotoxin components (i.e., LTB, STa, and STb) and were able to induce protective antitoxins in animals. Therefore, they could be included in a multivalent vaccine as a potent component to provide broad-spectrum protection against diarrhea caused by ETEC expressing various fimbriae. Though the immunogenicity of STa and STb was enhanced in the trivalent enterotoxin DNA vaccines constructed in this work, it was still weaker than that in the corresponding recombinant protein.
引文
[1]Svennerholm A M, Tobias J. Vaccines against enterotoxigenic Escherichia coli [J]. Expert Rev. Vaccines,2008,7(6):795-804.
[2]Wenneras C, Erling V. Prevalence of enterotoxigenic Escherichia coli-associated diarrhoea and carrier state in the developing world [J]. J. Health Popul. Nutr.,2004,22(4):370-382.
[3]尤建嵩,徐永平,牛冬燕,等.鸡卵黄抗体在防治家畜疾病中的应用[J].中国饲料,2003(19)：8-9,11.
[4]Nagy B, Fekete P Z. Enterotoxigenic Escherichia coli in veterinary medicine [J]. Int. J. Med. Microbiol.,2005,295(6-7):443-454.
[5]Gaastra W, Svennerholm A M. Colonization factors of human enterotoxigenic Escherichia coli (ETEC) [J]. Trends Microbiol.,1996,4(11):444-452.
[6]Dubreuil J D, Letellier A, Harel J. A recombinant Escherichia coli heat-stable enterotoxin b (STb) fusion protein eliciting neutralizing antibodies [J]. FEMS Immunol. Med. Microbiol.,1996,13(4): 317-323.
[7]Qadri F, Svennerholm A M, Faruque A S, et al. Enterotoxigenic Escherichia coli in developing countries:epidemiology, microbiology, clinical features, treatment, and prevention [J]. Clin. Microbiol. Rev.,2005,18(3):465-483.
[8]Allen K P, Randolph M M, Fleckenstein J M. Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains [J]. Infect. Immun.,2006,74(2):869-875.
[9]Berberov E M, Zhou Y, Francis D H, et al. Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins [J]. Infect. Immun.,2004,72(7):3914-3924.
[10]Zhang W, Berberov E M, Freeling J, et al. Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies [J]. Infect. Immun.,2006, 74(6):3107-3114.
[11]Sizemore D R, Roland K L, Ryan U S. Enterotoxigenic Escherichia coli virulence factors and vaccine approaches [J]. Expert Rev. Vaccines,2004,3(5):585-595.
[12]Cardenas L, Clements J D. Development of mucosal protection against the heat-stable enterotoxin (ST) of Escherichia coli by oral immunization with a genetic fusion delivered by a bacterial vector [J]. Infect. Immun.,1993,61(11):4629-4636.
[13]Clements J D. Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins [J]. Infect. Immun.,1990,58(5): 1159-1166.
[14]Lawrence R M, Huang P T, Glick J, et al. Expression of the cloned gene for enterotoxin STb of Escherichia coli [J]. Infect. Immun.,1990,58(4):970-977.
[15]Lowenadler B, Lake M, Elmblad A, et al. A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies [J]. FEMS Microbiol. Lett.,1991, 66(3):271-277.
[16]Rosales-Mendoza S, Alpuche-Solis A G, Soria-Guerra R E, et al. Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants [J]. Plant J.,2009,57(1):45-54.
[17]Saarilahti H T, Palva E T, Holmgren J, et al. Fusion of genes encoding Escherichia coli heat-stable enterotoxin and outer-membrane protein Ompc [J]. Infect. Immun.,1989,57(11):3663-3665.
[18]Sanchez J, Hirst T R, Uhlin B E. Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin [J]. Gene,1988,64(2):265-275.
[19]Sanchez J, Svennerholm A M, Holmgren J. Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit [J]. FEBS Lett.,1988,241(1-2): 110-114.
[20]Zhang W P, Zhang C X, Francis D H, et al. Genetic fusions of heat-labile (LT) and heat-stable (ST) toxoids of porcine enterotoxigenic Escherichia coli elicit neutralizing anti-LT and anti-STa antibodies [J]. Infect. Immun.,2010,78(1):316-325.
[21]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(05)：27-29.
[22]武冬梅,陈创夫,祝建波,等.含有MAR序列的STⅠ-STⅡ-LTB融合基因在苜蓿中的表达[J].扬州大学学报(农业与生命科学版),2007,No.110(02)：83-87.
[23]房海.大肠埃希氏菌[M].石家庄市：河北科学技术出版社,1997.
[24]Nataro J P, Kaper J B. Diarrheagenic Escherichia coli [J]. Clin. Microbiol. Rev.,1998,11(1): 142-201.
[25]Patel S K, Dotson J, Allen K P, et al. Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli [J]. Infect. Immun.,2004,72(3): 1786-1794.
[26]Betley M J, Miller V L, Mekalanos J J. Genetics of bacterial enterotoxins [J]. Annu. Rev. Microbiol.,1986,40:577-605.
[27]Clements J D, Finkelstein R A. Demonstration of shared and unique immunological determinants in enterotoxins from Vibrio cholerae and Escherichia coli [J]. Infect. Immun.,1978,22(3):709-713.
[28]Clements J D, Flint D C, Klipstein F A. Immunological and physicochemical characterization of heat-labile enterotoxins isolated from two strains of Escherichia coli [J]. Infect. Immun.,1982, 38(2):806-809.
[29]Klipstein F A, Engert R F, Clements J D. Immunization of rats with heat-labile enterotoxin provides uniform protection against heterologous serotypes of enterotoxigenic Escherichia coli [J]. Infect. Immun.,1981,32(3):1100-1104.
[30]Smith N W, Sack R B. Immunological cross-reactions of enterotoxins from Escherichia coli and Vibrio cholerae [J]. J. Infect. Dis.,1973,127(2):164-170.
[31]Gill D M, Richardson S H. Adenosine diphosphate-ribosylation of adenylate cyclase catalyzed by heat-labile enterotoxin of Escherichia coli:comparison with cholera toxin [J]. J. Infect. Dis.,1980, 141(1):64-70.
[32]Smith H W, Halls S. Transmissible nature of genetic factor in Escherichia coli that controls enterotoxin production [J]. J. Gen. Microbiol.,1968,52:319-323.
[33]Smith H W, Linggood M A. Transfer factors in Escherichia coli with particular regard to their incidence in enteropathogenic strains [J]. J. Gen. Microbiol.,1970,62(AUG):287-291.
[34]Gyles C, So M, Falkow S. The enterotoxin plasmids of Escherichia coli [J]. J. Infect. Dis.,1974, 130(1):40-49.
[35]Dallas W S, Gill D M, Falkow S. Cistrons encoding Escherichia coli heat-labile toxin [J]. J. Bacteriol.,1979,139(3):850-858.
[36]Yamamoto T, Tamura T, Ryoji M, et al. Sequence analysis of the heat-labile enterotoxin subunit B gene originating in human enterotoxigenic Escherichia coli [J]. J. Bacteriol.,1982,152(1): 506-509.
[37]王嘉福,冉雪琴,叶在荣,等.猪大肠杆菌热敏肠毒素A亚基基因的克隆及核苷酸序列分析[J].畜牧兽医学报,1997,28(06)：517-523.
[38]Gill D M, Clements J D, Robertson D C, et al. Subunit number and arrangement in Escherichia coli heat-labile enterotoxin [J]. Infect. Immun.,1981,33(3):677-682.
[39]高研,李卫芬,马国霞.产肠毒素大肠杆菌主要致病因子及其致病机理的研究进展[J].上海畜牧兽医通讯,2006(05)：2-5.
[40]Burgess M N, Bywater R J, Cowley C M, et al. Biological evaluation of a methanolsoluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves [J]. Infect. Immun., 1978,21(2):526-531.
[41]Dubreuil J D. Escherichia coli STb enterotoxin [J]. Microbiology-UK,1997,143:1783-1795.
[42]Reis M H L, Affonso M H T, Trabulsi L R, et al. Transfer of a CFA/I-ST plasmid promoted by a conjugative plasmid in a strain of Escherichia coli of serotype O128ac:H12 [J]. Infect. Immun., 1980,29(1):140-143.
[43]王玉炯,许崇波.大肠杆菌耐热性肠毒素(ST)的分子生物学研究进展[J].宁夏大学学报(自然科学版),2001,22(03)：331-336.
[44]Yoshimura S, Ikemura H, Watanabe H, et al. Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli [J]. Febs Letters,1985,181(1): 138-142.
[45]Shimonishi Y, Hidaka Y, Koizumi M, et al. Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli [J]. Febs Letters, 1987,215(1):165-170.
[46]Yamasaki S, Hidaka Y, Ito H, et al. Structural requirements for the spatial structure and toxicity of heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli [J]. Bull. Chem. Soc. Jpn.,1988, 61(5):1701-1706.
[47]Ozaki H, Sato T, Kubota H, et al. Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli. A putative binding site for a binding protein on rat intestinal epithelial cell membranes [J]. J. Biol. Chem.,1991,266(9):5934-5941.
[48]Akiyama Y, Kamitani S, Kusukawa N, et al. In vitro catalysis of oxidative folding of disulfide-bonded proteins by the Escherichia coli dsbA (ppfA) gene product [J]. J. Biol. Chem., 1992,267(31):22440-22445.
[49]Yamanaka H, Kameyama M, Baba T, et al. Maturation pathway of Escherichia coli heat-stable enterotoxin I:requirement of DsbA for disulfide bond formation [J]. J. Bacteriol.,1994,176(10): 2906-2913.
[50]Alderete J F, Robertson D C. Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli [J]. Infect. Immun., 1978,19(3):1021-1030.
[51]Giannella R A. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model [J]. Infect. Immun.,1976,14(1):95-99.
[52]Greenberg R N, Guerrant R L. Escherichia coli heat-stable enterotoxin [J]. Pharmacol. Ther.,1981, 13(3):507-531.
[53]Blanco M, Blanco J E, Gonzalez E A, et al. Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes:Relationship with toxic phenotypes [J]. J. Clin. Microbiol.,1997,35(11):2958-2963.
[54]Casey T A, Herring C J, Schneider R A, et al. Expression of heat-stable enterotoxin STb by adherent Escherichia coli is not sufficient to cause severe diarrhea in neonatal pigs [J]. Infect. Immun.,1998,66(3):1270-1272.
[55]Mainil J G, Daube G, Jacquemin E, et al. Virulence plasmids of enterotoxigenic Escherichia coli isolates from piglets [J]. Vet. Microbiol.,1998,62(4):291-301.
[56]Harnett N M, Gyles C L. Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin in bovine and porcine strains of enterotoxigenic Escherichia coli [J]. Am. J. Vet. Res., 1985,46(2):428-433.
[57]Echeverria P, Seriwatana J, Taylor D N, et al. Escherichia coli contains plasmids coding for heat-stable b, other enterotoxins, and antibiotic resistance [J]. Infect. Immun.,1985,48(3): 843-846.
[58]Lee C H, Hu S T, Swiatek P J, et al. Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene [J]. J. Bacteriol.,1985,162(2):615-620.
[59]Hu S T, Lee C H. Characterization of the transposon carrying the STⅡ gene of enterotoxigenic Escherichia coli [J]. Mol. Gen. Genet.,1988,214(3):490-495.
[60]Picken R N, Mazaitis A J, Maas W K, et al. Nucleotide sequence of the gene for heat-stable enterotoxin Ⅱ of Escherichia coli[J]. Infect. Immun.,1983,42(1):269-275.
[61]Kupersztoch Y M, Tachias K, Moomaw C R, et al. Secretion of a methanol-insoluble heat-stable enterotoxin (STB):energy-and sec-A dependent conversion of pre-STB to an intermediate indistinguishable from the extracellular toxin [J]. J. Bacteriol.,1990,172(5):2427-2432.
[62]Fujii Y, Hayashi M, Hitotsubashi S, et al. Purification and characterization of Escherichia coli heat-stable enterotoxin II [J]. J. Bacteriol.,1991,173(17):5516-5522.
[63]Dubreuil J D, Fairbrother J M, Lallier R, et al. Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain [J]. Infect. Immun.,1991,59(1):198-203.
[64]Rousset E, Harel J, Dubreuil J D. Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b [J]. Infect. Immun.,1998,66(12): 5650-5658.
[65]Labrie V, Beausoleil H E, Harel J, et al. Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants [J]. Microbiology-SGM,2001,147:3141-3148.
[66]Hitotsubashi S, Fujii Y, Yamanaka H, et al. Some properties of purified Escherichia coli heat-stable enterotoxin II [J]. Infect. Immun.,1992,60(11):4468-4474.
[67]Rask-Madsen J, Bukhave K. The role of prostaglandins in diarrhea, in Diarrhea:New Insights [M]. London:Janssen Pharmaceutical,1981.
[68]Fujii Y, Kondo Y, Okamoto K. Involvement of prostaglandin E2 synthesis in the intestinal secretory action of Escherichia coli heat-stable enterotoxin Ⅱ [J]. FEMS Microbiol. Lett.,1995, 130(2-3):259-265.
[69]Harville B A, Dreyfus L A. Involvement of 5-hydroxytryptamine and prostaglandin E2 in intestinal secretory action of Escherichia coli heat-stable enterotoxin B [J]. Infect. Immun.,1995,63(3): 745-750.
[70]Peterson J W, Whipp S C. Comparison of the mechanisms of action of cholera toxin and the heat-stable enterotoxins of Escherichia coli [J]. Infect. Immun.,1995,63(4):1452-1461.
[71]Eklund S, Jodal M, Lundgren O. The enteric nervous system participates in the secretory response to the heat stable enterotoxins of Escherichia coli in rats and cats [J]. Neuroscience,1985,14(2): 673-681.
[72]Hitotsubashi S, Akagi M, Saitou A, et al. Action of Escherichia coli heat-stable enterotoxin II on isolated sections of mouse ileum [J]. FEMS Microbiol. Lett.,1992,90(3):249-252.
[73]Dreyfus L A, Harville B, Howard D E, et al. Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB) [J]. Proc. Natl. Acad. Sci. U. S. A.,1993,90(8):3202-3206.
[74]Fujii Y, Nomura T, Yamanaka H, et al. Involvement of Ca2(+)-calmodulin-dependent protein kinase II in the intestinal secretory action of Escherichia coli heat-stable enterotoxin II [J]. Microbiol. Immunol.,1997,41(8):633-636.
[75]尤建嵩,牛冬燕,崔乃忠,等.大肠杆菌耐热肠毒素b及其致动物腹泻的机理[J].中国兽医科学,2006,36(10)：847-851.
[76]逄媛,沈志强,崔言顺,等.仔猪腹泻性大肠杆菌基因工程疫苗的研究进展[J].上海畜牧兽医通讯,2007(05)：2-3.
[77]Hasan U A, Abai A M, Harper D R, et al. Nucleic acid immunization:concepts and techniques associated with third generation vaccines [J]. J. Immunol. Methods,1999,229(1-2):1-22.
[78]Schultz J, Dollenmaier G, Moiling K. Update on antiviral DNA vaccine research (1998-2000) [J]. Intervirology,2000,43(4-6):197-217.
[79]Cohen J. Naked DNA Points Way to Vaccines [J]. Science,1993,259(5102):1690-1692.
[80]Donnelly J J, Friedman A, Martinez D, et al. Preclinical efficacy of a prototype DNA vaccine-enhanced protection against antigenic drift in influenza-virus [J]. Nat. Med.,1995,1(6): 583-587.
[81]刘国霞,姜宇,张斌,等.动物核酸疫苗研究进展[J].动物医学进展,2005(09)：34-36.
[82]王嘉福,冉雪琴,叶在荣,等.猪源大肠杆菌耐热肠毒素基因的分子克隆[J].畜牧兽医学报,1998,29(01)：60-65.
[83]Guzman-Verduzco L M, Kupersztoch Y M. Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit [J]. J. Bacteriol.,1987,169(11):5201-5208.
[84]Aitken R, Hirst T R. Recombinant enterotoxins as vaccines against Escherichia coli-mediated diarrhoea [J]. Vaccine,1993,11(2):227-233.
[85]张兆山,李淑琴,董自止,黄翠芬.不耐热肠毒素和耐热肠毒素基因的融合[J].中华微生物学和免疫学杂志,1994,14(04)：219-222.
[86]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(04)：439-443.
[87]徐兵,张兆山,李淑琴,等.大肠杆菌肠毒素的基因融合及其免疫原性的研究[J].中国生物化学与分子生物学报,1999,15(06)：903-906.
[88]武冬梅,祝建波,陈创夫,等.大肠杆菌肠毒素STⅠ、STⅡ、LTB融合基因植物表达载体的构建[J].西北农业学报,2006,15(05)：150-154.
[89]武冬梅,陈创夫,祝建波,等.含有MAR序列的STⅠ-STⅡ-LTB融合基因在苜蓿中的表达[J].扬州大学学报(农业与生命科学版),2007,28(02)：83-87.
[90]Dupasquier L, Schwager J, Flajnik M F. The Immune-System of Xenopus [J]. Annu. Rev. Immunol.,1989,7:251-275.
[91]Leslie G A, Clem L W. Phylogeny of Immunoglobulin Structure and Function.3. Immunoglobulins of Chicken [J]. J. Exp. Med.,1969,130(6):1337-1342.
[92]Klemperer F. Ueber natiirliche Immunitat und ihre Verwerthung fur die Immunisirungstherapie [J]. Archiv fur Experimentelle Pathologie und Pharmakologie,1893,31:356-382.
[93]Patterson R, Weigle W O, Youngner J S, et al. Antibody Production and Transfer to Egg Yolk in Chickens [J]. J. Immunol.,1962,89(2):272-278.
[94]Loeken M R, Roth T F. Analysis of Maternal Igg Subpopulations Which Are Transported into the Chicken Oocyte [J]. Immunology,1983,49(1):21-28.
[95]Leslie G A, Martin L N. Studies on the secretory immunologic system of fowl.3. Serum and secretory IgA of the chicken [J]. J. Immunol.,1973,110(1):1-9.
[96]彭玉麟,呙于明.鸡卵黄免疫球蛋自在畜禽生产中的应用前景[J].中国饲料,2002(06)：26-28.
[97]Schade R,张小莺,郑礼.lgY技术及其医药应用：理论基础[J].中国药理学通报,2004(05)：491-495.
[98]Yamamoto H, Watanabe H, Sato G, et al. Identification of Immunoglobulins in Chicken Eggs and Their Antibody Activity [J]. Jpn. J. Vet. Res.,1975,23(4):131-135.
[99]Woolley J A, Landon J. Comparison of Antibody-Production to Human Interleukin-6 (Ⅱ-6) by Sheep and Chickens [J]. J. Immunol. Methods,1995,178(2):253-265.
[100]Ambrosius H. Developmental aspects of immunoglobulins and antibodies. [J]. ALTEX,1996, 13(Suppl.1):10-14.
[101]Carlander D. Avian IgY Antibody. In Vitro and In Vivo [D]. University of Uppsala:Uppsala, Sweden,2002.
[102]Warr G W, Magor K. E, Higgins D A. IgY-Clues to the Origins of Modern Antibodies [J]. Immunol. Today,1995,16(8):392-398.
[103]Davalos-Pantoja L, Ortega-Vinuesa J L, Bastos-Gonzalez D, et al. A comparative study between the adsorption of IgY and IgG on latex particles [J]. J. Biomater. Sci.-Polymer Edition,2000, 11(6):657-673.
[104]Schade R, Calzado E G, Sarmiento R, et al. Chicken egg yolk antibodies (IgY-technology):A review of progress in production and use in research and human and veterinary medicine [J]. Atla-Altern. to Lab. Anim.,2005,33(2):129-154.
[105]Shimizu M, Nagashima H, Sano K, et al. Molecular-stability of chicken and rabbit immunoglobulin-G [J]. Biosci. Biotechnol. Biochem.,1992,56(2):270-274.
[106]Schmidt P, Erhard M H, Schams D, et al. Chicken egg antibodies for immunohistochemical labeling of growth-hormone and prolactin in bovine pituitary-gland [J]. J. Histochem. Cytochem., 1993,41(9):1441-1446.
[107]王炯,龚春梅,赵树栋,等.鸡卵抗体(1gY)理化特性的研究[J].中国生物制品学杂志,1997(03)：140-143.
[108]龙中儿,钟青萍,朱跃科,等.抗轮状病毒卵黄免疫球蛋白的制备及其活性[J].南昌大学学报(理科版),1997(02)：30-35,51.
[109]郭立君,李春晖,赵锋,等.抗轮状病毒lgY的研制[J].中国生物制品学杂志,2001(01)：24-26.
[110]Larsson A, Balow R M, Lindahl T L, et al. Chicken Antibodies-Taking Advantage of Evolution-a Review [J]. Poult. Sci.,1993,72(10):1807-1812.
[111]Hatta H, Tsuda K, Akachi S, et al. Productivity and some properties of egg-yolk antibody (IgY) against human rotavirus compared with rabbit IgG [J]. Biosci. Biotechnol. Biochem.,1993,57(3): 450-454.
[112]Lee K A, Chang S K, Lee Y J, et al. Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution [J]. J. Biochem. Molecul. Bio.,2002,35(5):488-493.
[113]Losch U, Schranner I, Wanke R, et al. The Chicken Egg, an Antibody Source [J]. J. Vet. Med. Series B-Infect. Dis. Vet. Public Health,1986,33(8):609-619.
[114]Hatta H, Tsuda K, Akachi S, et al. Oral passive-immunization effect of anti-human rotavirus IgY and its behavior against proteolytic-enzymes [J]. Biosci. Biotechnol. Biochem.,1993,57(7): 1077-1081.
[115]Russell W M S, Burch, R.L. The Principles of Humane Experimental Technique [M]. London: Methuen.238,1959.
[116]易全茂,谢艳霞.鸡卵黄抗体研究进展[J].动物科学与动物医学,2004(02)：44-45,33.
[117]程学慧,彭健,詹志春.新型饲料添加剂——卵黄抗体研究进展[J].饲料研究,2003(05)：19-21.
[118]Mine Y, Kovacs-Nolan J. Chicken egg yolk antibodies as therapeutics in enteric infectious disease:a review. [J]. J. Med. Food 2002,5(3):159-169.
[119]钟青萍,杨荣鉴,熊勇华,等.鸡卵黄免疫球蛋白研究动态[J].生命科学,1998(05)：252-254.
[120]Schmidt P, Wiedemann V, Kuhlmann R, et al. Chicken egg antibodies for prophylaxis and therapy of infectious intestinal-diseases.2. In vitro studies on gastric and enteric digestion of egg-yolk antibodies specific against pathogenic Escherichia coli strains [J]. J. Vet. Med. Series B- Infect. Dis. Vet. Public Health,1989,36(8):619-628.
[121]Wiedemann V, Kuhlmann R, Schmidt P, et al. Chicken egg antibodies for prophylaxis and therapy of infectious intestinal-diseases.3. In vivo tenacity test in piglets with artificial jejunal fistula [J]. J. Vet. Med. Series B-Infect. Dis. Vet. Public Health,1990,37(3):163-172.
[122]Yokoyama H, Peralta R C, Diaz R, et al. Passive protective effect of chicken egg-yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets [J]. Infect. Immun.,1992,60(3):998-1007.
[123]徐福洲,王金洛,杨兵,等.抗猪产肠毒素大肠埃希氏菌卵黄抗体的研制及攻毒治疗试验[J].华北农学报,2002(04)：108-112.
[124]高云英,赵发苗,张金良,等.鸡抗猪大肠杆菌高免卵黄抗体的研制与应用[J].畜牧兽医杂志,2003(01)：6-8.
[125]Erhard M H, Bergmann J, Renner M, et al. Prophylactic effect of specific egg yolk antibodies in diarrhea of weaned piglets caused by Escherichia coli K.88 (F4) [J]. Journal of Veterinary Medicine Series a-Zentralblatt Fur Veterinarmedizin Reihe a-Physiology Pathology Clinical Medicine,1996,43(4):217-223.
[126]Kweon C H, Kwon B J, Woo S R, et al. Immunoprophylactic effect of chicken egg yolk immunoglobulin (Ig Y) against porcine epidemic diarrhea virus (PEDV) in piglets [J]. J. Vet. Med. Sci.,2000,62(9):961-964.
[127]Erhard M H, Kellner J, Eichelberger J, et al. New aspects in oral immunoprophylaxis for the prevention of infectious diarrhea of newborn calves —A field-study with specific egg antibodies [J]. Berl. Munch. Tierarztl. Wochenschr.,1993,106(11):383-387.
[128]Ikemori Y, Kuroki M, Peralta R C, et al. Protection of neonatal calves against fatal enteric colibacillosis by administration of egg-yolk powder from hens immunized with k99-piliated enterotoxigenic Escherichia coli [J]. Am. J. Vet. Res.,1992,53(11):2005-2008.
[129]Kuroki M, Ikemori Y, Yokoyama H, et al. Passive protection against bovine rotavirus-induced diarrhea in murine model by specific immunoglobulins from chicken egg-yolk [J]. Vet. Microbiol., 1993,37(1-2):135-146.
[130]Yokoyama H, Peralta R C, Umeda K, et al. Prevention of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk Salmonella-specific antibodies [J]. Am. J. Vet. Res., 1998,59(4):416-420.
[131]Hirai K., Arimitsu H, Umeda K, et al. Passive Oral Immunization by Egg Yolk Immunoglobulin (IgY) to Vibrio cholerae Effectively Prevents Cholera [J]. Acta Med. Okayama,2010,64(3): 163-170.
[132]LeClaire R D, Hunt R E, Bavari S. Protection against bacterial superantigen staphylococcal enterotoxin B by passive vaccination [J]. Infect. Immun.,2002,70(5):2278-2281.
[133]胡思顺.家畜DNA疫苗免疫最新进展及应用前景[J].《国外医学》预防、诊断、治疗用生物制品分册,2002,25(3)：107-110.
[134]Kirman J R, Turon T, Su H, et al. Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen 85A [J]. Infect. Immun.,2003, 71:575-579.
[135]Deliyannis G, Boyle J S, Brady J L, et al. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge [J]. Proc. Natl. Acad. Sci. U. S. A.,2000,97(12): 6676-6680.
[136]Baldwin S L, D'Souza C D, Orme 1 M, et al. Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A [J]. Tuber. LungDis.,1999,79:251-259.
[137]Leong J, Vinal A C, Dallas W S. Nucleotide-sequence comparison between heat-labile toxin b-subunit cistrons from Escherichia coli of human and porcine origin [J]. Infect. Immun.,1985, 48(1):73-77.
[138]So M, Mccarthy B J. Nucleotide-sequence of the bacterial transposon tn 1681 encoding a heat-stable (st) toxin and its identification in entero-toxigenic Escherichia coli strains [J]. Proc. Natl. Acad. Sci. U. S. A.-Biol. Sci.,1980,77(7):4011-4015.
[139]Lee C H, Moseley S L, Moon H W, et al. Characterization of the gene encoding heat-stable toxin-ii and preliminary molecular epidemiological-studies of enterotoxigenic Escherichia coli heat-stable toxin-II producers [J]. Infect. Immun.,1983,42(1):264-268.
[140]卢圣栋.现代分子生物学实验技术[M].第二版.北京：中国协和医科大学出版社,1999.
[141]汪家政,范明.蛋白质技术手册[M].北京：科学出版社,2000.
[142]Kunkel S L, Robertson D C. Purification and chemical characterization of the heat-labile enterotoxin produced by enterotoxigenic Escherichia coli [J]. Infect. Immun.,1979,25(2): 586-596.
[143]Saeed A M K, Sriranganathan N, Cosand W, et al. Purification and characterization of heat-stable enterotoxin from bovine enterotoxigenic Escherichia coli [J]. Infect. Immun.,1983,40(2): 701-707.
[144]Mundell D H, Anselmo C R, Wishnow R M. Factors influencing heat-labile Escherichia coli enterotoxin activity [J]. Infect. Immun.,1976,14(2):383-388.
[145]Staples S J, Asher S E, Giannella R A. Purification and characterization of heat-stable enterotoxin produced by a strain of E. coli pathogenic for man [J]. J. Biol. Chem.,1980,10:4716-4721.
[146]Akita E M, Nakai S. Immunoglobulins from egg yolk:isolation and purification [J]. J. Food Sci., 1992,57(3):629-634.
[147]Maya D C, Vasantha B M, Vijayan L A, et al. An improved method for isolation of anti-viper venom antibodies from chicken egg yolk [J]. J. Biochem. Biophys. Meth.,2002,51(2):129-138.
[148]Whipp S C. Assay of enterotoxigenic Escherichia coli heat-stable toxin b in rats and mice [J]. Infect. Immun.,1990,58:930-934.
[149]Wu C M, Chung T C. Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein [J]. FEMS Immunol. Med. Microbiol.,2007,50(3):354-65.
[150]李建荣,于涟,黄耀伟,等.免疫刺激复合物(ISCOM)介导的传染性法氏囊病病毒多聚蛋白基因免疫的研究[J].病毒学报,2001,17(4)：341-348.
[2]Wenneras C, Erling V. Prevalence of enterotoxigenic Escherichia coli-associated diarrhoea and carrier state in the developing world [J]. J. Health Popul. Nutr.,2004,22(4):370-382.
[3]尤建嵩,徐永平,牛冬燕,等.鸡卵黄抗体在防治家畜疾病中的应用[J].中国饲料,2003(19)：8-9,11.
[4]Nagy B, Fekete P Z. Enterotoxigenic Escherichia coli in veterinary medicine [J]. Int. J. Med. Microbiol.,2005,295(6-7):443-454.
[5]Gaastra W, Svennerholm A M. Colonization factors of human enterotoxigenic Escherichia coli (ETEC) [J]. Trends Microbiol.,1996,4(11):444-452.
[6]Dubreuil J D, Letellier A, Harel J. A recombinant Escherichia coli heat-stable enterotoxin b (STb) fusion protein eliciting neutralizing antibodies [J]. FEMS Immunol. Med. Microbiol.,1996,13(4): 317-323.
[7]Qadri F, Svennerholm A M, Faruque A S, et al. Enterotoxigenic Escherichia coli in developing countries:epidemiology, microbiology, clinical features, treatment, and prevention [J]. Clin. Microbiol. Rev.,2005,18(3):465-483.
[8]Allen K P, Randolph M M, Fleckenstein J M. Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains [J]. Infect. Immun.,2006,74(2):869-875.
[9]Berberov E M, Zhou Y, Francis D H, et al. Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins [J]. Infect. Immun.,2004,72(7):3914-3924.
[10]Zhang W, Berberov E M, Freeling J, et al. Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies [J]. Infect. Immun.,2006, 74(6):3107-3114.
[11]Sizemore D R, Roland K L, Ryan U S. Enterotoxigenic Escherichia coli virulence factors and vaccine approaches [J]. Expert Rev. Vaccines,2004,3(5):585-595.
[12]Cardenas L, Clements J D. Development of mucosal protection against the heat-stable enterotoxin (ST) of Escherichia coli by oral immunization with a genetic fusion delivered by a bacterial vector [J]. Infect. Immun.,1993,61(11):4629-4636.
[13]Clements J D. Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins [J]. Infect. Immun.,1990,58(5): 1159-1166.
[14]Lawrence R M, Huang P T, Glick J, et al. Expression of the cloned gene for enterotoxin STb of Escherichia coli [J]. Infect. Immun.,1990,58(4):970-977.
[15]Lowenadler B, Lake M, Elmblad A, et al. A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies [J]. FEMS Microbiol. Lett.,1991, 66(3):271-277.
[16]Rosales-Mendoza S, Alpuche-Solis A G, Soria-Guerra R E, et al. Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants [J]. Plant J.,2009,57(1):45-54.
[17]Saarilahti H T, Palva E T, Holmgren J, et al. Fusion of genes encoding Escherichia coli heat-stable enterotoxin and outer-membrane protein Ompc [J]. Infect. Immun.,1989,57(11):3663-3665.
[18]Sanchez J, Hirst T R, Uhlin B E. Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin [J]. Gene,1988,64(2):265-275.
[19]Sanchez J, Svennerholm A M, Holmgren J. Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit [J]. FEBS Lett.,1988,241(1-2): 110-114.
[20]Zhang W P, Zhang C X, Francis D H, et al. Genetic fusions of heat-labile (LT) and heat-stable (ST) toxoids of porcine enterotoxigenic Escherichia coli elicit neutralizing anti-LT and anti-STa antibodies [J]. Infect. Immun.,2010,78(1):316-325.
[21]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(05)：27-29.
[22]武冬梅,陈创夫,祝建波,等.含有MAR序列的STⅠ-STⅡ-LTB融合基因在苜蓿中的表达[J].扬州大学学报(农业与生命科学版),2007,No.110(02)：83-87.
[23]房海.大肠埃希氏菌[M].石家庄市：河北科学技术出版社,1997.
[24]Nataro J P, Kaper J B. Diarrheagenic Escherichia coli [J]. Clin. Microbiol. Rev.,1998,11(1): 142-201.
[25]Patel S K, Dotson J, Allen K P, et al. Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli [J]. Infect. Immun.,2004,72(3): 1786-1794.
[26]Betley M J, Miller V L, Mekalanos J J. Genetics of bacterial enterotoxins [J]. Annu. Rev. Microbiol.,1986,40:577-605.
[27]Clements J D, Finkelstein R A. Demonstration of shared and unique immunological determinants in enterotoxins from Vibrio cholerae and Escherichia coli [J]. Infect. Immun.,1978,22(3):709-713.
[28]Clements J D, Flint D C, Klipstein F A. Immunological and physicochemical characterization of heat-labile enterotoxins isolated from two strains of Escherichia coli [J]. Infect. Immun.,1982, 38(2):806-809.
[29]Klipstein F A, Engert R F, Clements J D. Immunization of rats with heat-labile enterotoxin provides uniform protection against heterologous serotypes of enterotoxigenic Escherichia coli [J]. Infect. Immun.,1981,32(3):1100-1104.
[30]Smith N W, Sack R B. Immunological cross-reactions of enterotoxins from Escherichia coli and Vibrio cholerae [J]. J. Infect. Dis.,1973,127(2):164-170.
[31]Gill D M, Richardson S H. Adenosine diphosphate-ribosylation of adenylate cyclase catalyzed by heat-labile enterotoxin of Escherichia coli:comparison with cholera toxin [J]. J. Infect. Dis.,1980, 141(1):64-70.
[32]Smith H W, Halls S. Transmissible nature of genetic factor in Escherichia coli that controls enterotoxin production [J]. J. Gen. Microbiol.,1968,52:319-323.
[33]Smith H W, Linggood M A. Transfer factors in Escherichia coli with particular regard to their incidence in enteropathogenic strains [J]. J. Gen. Microbiol.,1970,62(AUG):287-291.
[34]Gyles C, So M, Falkow S. The enterotoxin plasmids of Escherichia coli [J]. J. Infect. Dis.,1974, 130(1):40-49.
[35]Dallas W S, Gill D M, Falkow S. Cistrons encoding Escherichia coli heat-labile toxin [J]. J. Bacteriol.,1979,139(3):850-858.
[36]Yamamoto T, Tamura T, Ryoji M, et al. Sequence analysis of the heat-labile enterotoxin subunit B gene originating in human enterotoxigenic Escherichia coli [J]. J. Bacteriol.,1982,152(1): 506-509.
[37]王嘉福,冉雪琴,叶在荣,等.猪大肠杆菌热敏肠毒素A亚基基因的克隆及核苷酸序列分析[J].畜牧兽医学报,1997,28(06)：517-523.
[38]Gill D M, Clements J D, Robertson D C, et al. Subunit number and arrangement in Escherichia coli heat-labile enterotoxin [J]. Infect. Immun.,1981,33(3):677-682.
[39]高研,李卫芬,马国霞.产肠毒素大肠杆菌主要致病因子及其致病机理的研究进展[J].上海畜牧兽医通讯,2006(05)：2-5.
[40]Burgess M N, Bywater R J, Cowley C M, et al. Biological evaluation of a methanolsoluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves [J]. Infect. Immun., 1978,21(2):526-531.
[41]Dubreuil J D. Escherichia coli STb enterotoxin [J]. Microbiology-UK,1997,143:1783-1795.
[42]Reis M H L, Affonso M H T, Trabulsi L R, et al. Transfer of a CFA/I-ST plasmid promoted by a conjugative plasmid in a strain of Escherichia coli of serotype O128ac:H12 [J]. Infect. Immun., 1980,29(1):140-143.
[43]王玉炯,许崇波.大肠杆菌耐热性肠毒素(ST)的分子生物学研究进展[J].宁夏大学学报(自然科学版),2001,22(03)：331-336.
[44]Yoshimura S, Ikemura H, Watanabe H, et al. Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli [J]. Febs Letters,1985,181(1): 138-142.
[45]Shimonishi Y, Hidaka Y, Koizumi M, et al. Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli [J]. Febs Letters, 1987,215(1):165-170.
[46]Yamasaki S, Hidaka Y, Ito H, et al. Structural requirements for the spatial structure and toxicity of heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli [J]. Bull. Chem. Soc. Jpn.,1988, 61(5):1701-1706.
[47]Ozaki H, Sato T, Kubota H, et al. Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli. A putative binding site for a binding protein on rat intestinal epithelial cell membranes [J]. J. Biol. Chem.,1991,266(9):5934-5941.
[48]Akiyama Y, Kamitani S, Kusukawa N, et al. In vitro catalysis of oxidative folding of disulfide-bonded proteins by the Escherichia coli dsbA (ppfA) gene product [J]. J. Biol. Chem., 1992,267(31):22440-22445.
[49]Yamanaka H, Kameyama M, Baba T, et al. Maturation pathway of Escherichia coli heat-stable enterotoxin I:requirement of DsbA for disulfide bond formation [J]. J. Bacteriol.,1994,176(10): 2906-2913.
[50]Alderete J F, Robertson D C. Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli [J]. Infect. Immun., 1978,19(3):1021-1030.
[51]Giannella R A. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model [J]. Infect. Immun.,1976,14(1):95-99.
[52]Greenberg R N, Guerrant R L. Escherichia coli heat-stable enterotoxin [J]. Pharmacol. Ther.,1981, 13(3):507-531.
[53]Blanco M, Blanco J E, Gonzalez E A, et al. Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes:Relationship with toxic phenotypes [J]. J. Clin. Microbiol.,1997,35(11):2958-2963.
[54]Casey T A, Herring C J, Schneider R A, et al. Expression of heat-stable enterotoxin STb by adherent Escherichia coli is not sufficient to cause severe diarrhea in neonatal pigs [J]. Infect. Immun.,1998,66(3):1270-1272.
[55]Mainil J G, Daube G, Jacquemin E, et al. Virulence plasmids of enterotoxigenic Escherichia coli isolates from piglets [J]. Vet. Microbiol.,1998,62(4):291-301.
[56]Harnett N M, Gyles C L. Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin in bovine and porcine strains of enterotoxigenic Escherichia coli [J]. Am. J. Vet. Res., 1985,46(2):428-433.
[57]Echeverria P, Seriwatana J, Taylor D N, et al. Escherichia coli contains plasmids coding for heat-stable b, other enterotoxins, and antibiotic resistance [J]. Infect. Immun.,1985,48(3): 843-846.
[58]Lee C H, Hu S T, Swiatek P J, et al. Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene [J]. J. Bacteriol.,1985,162(2):615-620.
[59]Hu S T, Lee C H. Characterization of the transposon carrying the STⅡ gene of enterotoxigenic Escherichia coli [J]. Mol. Gen. Genet.,1988,214(3):490-495.
[60]Picken R N, Mazaitis A J, Maas W K, et al. Nucleotide sequence of the gene for heat-stable enterotoxin Ⅱ of Escherichia coli[J]. Infect. Immun.,1983,42(1):269-275.
[61]Kupersztoch Y M, Tachias K, Moomaw C R, et al. Secretion of a methanol-insoluble heat-stable enterotoxin (STB):energy-and sec-A dependent conversion of pre-STB to an intermediate indistinguishable from the extracellular toxin [J]. J. Bacteriol.,1990,172(5):2427-2432.
[62]Fujii Y, Hayashi M, Hitotsubashi S, et al. Purification and characterization of Escherichia coli heat-stable enterotoxin II [J]. J. Bacteriol.,1991,173(17):5516-5522.
[63]Dubreuil J D, Fairbrother J M, Lallier R, et al. Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain [J]. Infect. Immun.,1991,59(1):198-203.
[64]Rousset E, Harel J, Dubreuil J D. Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b [J]. Infect. Immun.,1998,66(12): 5650-5658.
[65]Labrie V, Beausoleil H E, Harel J, et al. Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants [J]. Microbiology-SGM,2001,147:3141-3148.
[66]Hitotsubashi S, Fujii Y, Yamanaka H, et al. Some properties of purified Escherichia coli heat-stable enterotoxin II [J]. Infect. Immun.,1992,60(11):4468-4474.
[67]Rask-Madsen J, Bukhave K. The role of prostaglandins in diarrhea, in Diarrhea:New Insights [M]. London:Janssen Pharmaceutical,1981.
[68]Fujii Y, Kondo Y, Okamoto K. Involvement of prostaglandin E2 synthesis in the intestinal secretory action of Escherichia coli heat-stable enterotoxin Ⅱ [J]. FEMS Microbiol. Lett.,1995, 130(2-3):259-265.
[69]Harville B A, Dreyfus L A. Involvement of 5-hydroxytryptamine and prostaglandin E2 in intestinal secretory action of Escherichia coli heat-stable enterotoxin B [J]. Infect. Immun.,1995,63(3): 745-750.
[70]Peterson J W, Whipp S C. Comparison of the mechanisms of action of cholera toxin and the heat-stable enterotoxins of Escherichia coli [J]. Infect. Immun.,1995,63(4):1452-1461.
[71]Eklund S, Jodal M, Lundgren O. The enteric nervous system participates in the secretory response to the heat stable enterotoxins of Escherichia coli in rats and cats [J]. Neuroscience,1985,14(2): 673-681.
[72]Hitotsubashi S, Akagi M, Saitou A, et al. Action of Escherichia coli heat-stable enterotoxin II on isolated sections of mouse ileum [J]. FEMS Microbiol. Lett.,1992,90(3):249-252.
[73]Dreyfus L A, Harville B, Howard D E, et al. Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB) [J]. Proc. Natl. Acad. Sci. U. S. A.,1993,90(8):3202-3206.
[74]Fujii Y, Nomura T, Yamanaka H, et al. Involvement of Ca2(+)-calmodulin-dependent protein kinase II in the intestinal secretory action of Escherichia coli heat-stable enterotoxin II [J]. Microbiol. Immunol.,1997,41(8):633-636.
[75]尤建嵩,牛冬燕,崔乃忠,等.大肠杆菌耐热肠毒素b及其致动物腹泻的机理[J].中国兽医科学,2006,36(10)：847-851.
[76]逄媛,沈志强,崔言顺,等.仔猪腹泻性大肠杆菌基因工程疫苗的研究进展[J].上海畜牧兽医通讯,2007(05)：2-3.
[77]Hasan U A, Abai A M, Harper D R, et al. Nucleic acid immunization:concepts and techniques associated with third generation vaccines [J]. J. Immunol. Methods,1999,229(1-2):1-22.
[78]Schultz J, Dollenmaier G, Moiling K. Update on antiviral DNA vaccine research (1998-2000) [J]. Intervirology,2000,43(4-6):197-217.
[79]Cohen J. Naked DNA Points Way to Vaccines [J]. Science,1993,259(5102):1690-1692.
[80]Donnelly J J, Friedman A, Martinez D, et al. Preclinical efficacy of a prototype DNA vaccine-enhanced protection against antigenic drift in influenza-virus [J]. Nat. Med.,1995,1(6): 583-587.
[81]刘国霞,姜宇,张斌,等.动物核酸疫苗研究进展[J].动物医学进展,2005(09)：34-36.
[82]王嘉福,冉雪琴,叶在荣,等.猪源大肠杆菌耐热肠毒素基因的分子克隆[J].畜牧兽医学报,1998,29(01)：60-65.
[83]Guzman-Verduzco L M, Kupersztoch Y M. Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit [J]. J. Bacteriol.,1987,169(11):5201-5208.
[84]Aitken R, Hirst T R. Recombinant enterotoxins as vaccines against Escherichia coli-mediated diarrhoea [J]. Vaccine,1993,11(2):227-233.
[85]张兆山,李淑琴,董自止,黄翠芬.不耐热肠毒素和耐热肠毒素基因的融合[J].中华微生物学和免疫学杂志,1994,14(04)：219-222.
[86]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(04)：439-443.
[87]徐兵,张兆山,李淑琴,等.大肠杆菌肠毒素的基因融合及其免疫原性的研究[J].中国生物化学与分子生物学报,1999,15(06)：903-906.
[88]武冬梅,祝建波,陈创夫,等.大肠杆菌肠毒素STⅠ、STⅡ、LTB融合基因植物表达载体的构建[J].西北农业学报,2006,15(05)：150-154.
[89]武冬梅,陈创夫,祝建波,等.含有MAR序列的STⅠ-STⅡ-LTB融合基因在苜蓿中的表达[J].扬州大学学报(农业与生命科学版),2007,28(02)：83-87.
[90]Dupasquier L, Schwager J, Flajnik M F. The Immune-System of Xenopus [J]. Annu. Rev. Immunol.,1989,7:251-275.
[91]Leslie G A, Clem L W. Phylogeny of Immunoglobulin Structure and Function.3. Immunoglobulins of Chicken [J]. J. Exp. Med.,1969,130(6):1337-1342.
[92]Klemperer F. Ueber natiirliche Immunitat und ihre Verwerthung fur die Immunisirungstherapie [J]. Archiv fur Experimentelle Pathologie und Pharmakologie,1893,31:356-382.
[93]Patterson R, Weigle W O, Youngner J S, et al. Antibody Production and Transfer to Egg Yolk in Chickens [J]. J. Immunol.,1962,89(2):272-278.
[94]Loeken M R, Roth T F. Analysis of Maternal Igg Subpopulations Which Are Transported into the Chicken Oocyte [J]. Immunology,1983,49(1):21-28.
[95]Leslie G A, Martin L N. Studies on the secretory immunologic system of fowl.3. Serum and secretory IgA of the chicken [J]. J. Immunol.,1973,110(1):1-9.
[96]彭玉麟,呙于明.鸡卵黄免疫球蛋自在畜禽生产中的应用前景[J].中国饲料,2002(06)：26-28.
[97]Schade R,张小莺,郑礼.lgY技术及其医药应用：理论基础[J].中国药理学通报,2004(05)：491-495.
[98]Yamamoto H, Watanabe H, Sato G, et al. Identification of Immunoglobulins in Chicken Eggs and Their Antibody Activity [J]. Jpn. J. Vet. Res.,1975,23(4):131-135.
[99]Woolley J A, Landon J. Comparison of Antibody-Production to Human Interleukin-6 (Ⅱ-6) by Sheep and Chickens [J]. J. Immunol. Methods,1995,178(2):253-265.
[100]Ambrosius H. Developmental aspects of immunoglobulins and antibodies. [J]. ALTEX,1996, 13(Suppl.1):10-14.
[101]Carlander D. Avian IgY Antibody. In Vitro and In Vivo [D]. University of Uppsala:Uppsala, Sweden,2002.
[102]Warr G W, Magor K. E, Higgins D A. IgY-Clues to the Origins of Modern Antibodies [J]. Immunol. Today,1995,16(8):392-398.
[103]Davalos-Pantoja L, Ortega-Vinuesa J L, Bastos-Gonzalez D, et al. A comparative study between the adsorption of IgY and IgG on latex particles [J]. J. Biomater. Sci.-Polymer Edition,2000, 11(6):657-673.
[104]Schade R, Calzado E G, Sarmiento R, et al. Chicken egg yolk antibodies (IgY-technology):A review of progress in production and use in research and human and veterinary medicine [J]. Atla-Altern. to Lab. Anim.,2005,33(2):129-154.
[105]Shimizu M, Nagashima H, Sano K, et al. Molecular-stability of chicken and rabbit immunoglobulin-G [J]. Biosci. Biotechnol. Biochem.,1992,56(2):270-274.
[106]Schmidt P, Erhard M H, Schams D, et al. Chicken egg antibodies for immunohistochemical labeling of growth-hormone and prolactin in bovine pituitary-gland [J]. J. Histochem. Cytochem., 1993,41(9):1441-1446.
[107]王炯,龚春梅,赵树栋,等.鸡卵抗体(1gY)理化特性的研究[J].中国生物制品学杂志,1997(03)：140-143.
[108]龙中儿,钟青萍,朱跃科,等.抗轮状病毒卵黄免疫球蛋白的制备及其活性[J].南昌大学学报(理科版),1997(02)：30-35,51.
[109]郭立君,李春晖,赵锋,等.抗轮状病毒lgY的研制[J].中国生物制品学杂志,2001(01)：24-26.
[110]Larsson A, Balow R M, Lindahl T L, et al. Chicken Antibodies-Taking Advantage of Evolution-a Review [J]. Poult. Sci.,1993,72(10):1807-1812.
[111]Hatta H, Tsuda K, Akachi S, et al. Productivity and some properties of egg-yolk antibody (IgY) against human rotavirus compared with rabbit IgG [J]. Biosci. Biotechnol. Biochem.,1993,57(3): 450-454.
[112]Lee K A, Chang S K, Lee Y J, et al. Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution [J]. J. Biochem. Molecul. Bio.,2002,35(5):488-493.
[113]Losch U, Schranner I, Wanke R, et al. The Chicken Egg, an Antibody Source [J]. J. Vet. Med. Series B-Infect. Dis. Vet. Public Health,1986,33(8):609-619.
[114]Hatta H, Tsuda K, Akachi S, et al. Oral passive-immunization effect of anti-human rotavirus IgY and its behavior against proteolytic-enzymes [J]. Biosci. Biotechnol. Biochem.,1993,57(7): 1077-1081.
[115]Russell W M S, Burch, R.L. The Principles of Humane Experimental Technique [M]. London: Methuen.238,1959.
[116]易全茂,谢艳霞.鸡卵黄抗体研究进展[J].动物科学与动物医学,2004(02)：44-45,33.
[117]程学慧,彭健,詹志春.新型饲料添加剂——卵黄抗体研究进展[J].饲料研究,2003(05)：19-21.
[118]Mine Y, Kovacs-Nolan J. Chicken egg yolk antibodies as therapeutics in enteric infectious disease:a review. [J]. J. Med. Food 2002,5(3):159-169.
[119]钟青萍,杨荣鉴,熊勇华,等.鸡卵黄免疫球蛋白研究动态[J].生命科学,1998(05)：252-254.
[120]Schmidt P, Wiedemann V, Kuhlmann R, et al. Chicken egg antibodies for prophylaxis and therapy of infectious intestinal-diseases.2. In vitro studies on gastric and enteric digestion of egg-yolk antibodies specific against pathogenic Escherichia coli strains [J]. J. Vet. Med. Series B- Infect. Dis. Vet. Public Health,1989,36(8):619-628.
[121]Wiedemann V, Kuhlmann R, Schmidt P, et al. Chicken egg antibodies for prophylaxis and therapy of infectious intestinal-diseases.3. In vivo tenacity test in piglets with artificial jejunal fistula [J]. J. Vet. Med. Series B-Infect. Dis. Vet. Public Health,1990,37(3):163-172.
[122]Yokoyama H, Peralta R C, Diaz R, et al. Passive protective effect of chicken egg-yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets [J]. Infect. Immun.,1992,60(3):998-1007.
[123]徐福洲,王金洛,杨兵,等.抗猪产肠毒素大肠埃希氏菌卵黄抗体的研制及攻毒治疗试验[J].华北农学报,2002(04)：108-112.
[124]高云英,赵发苗,张金良,等.鸡抗猪大肠杆菌高免卵黄抗体的研制与应用[J].畜牧兽医杂志,2003(01)：6-8.
[125]Erhard M H, Bergmann J, Renner M, et al. Prophylactic effect of specific egg yolk antibodies in diarrhea of weaned piglets caused by Escherichia coli K.88 (F4) [J]. Journal of Veterinary Medicine Series a-Zentralblatt Fur Veterinarmedizin Reihe a-Physiology Pathology Clinical Medicine,1996,43(4):217-223.
[126]Kweon C H, Kwon B J, Woo S R, et al. Immunoprophylactic effect of chicken egg yolk immunoglobulin (Ig Y) against porcine epidemic diarrhea virus (PEDV) in piglets [J]. J. Vet. Med. Sci.,2000,62(9):961-964.
[127]Erhard M H, Kellner J, Eichelberger J, et al. New aspects in oral immunoprophylaxis for the prevention of infectious diarrhea of newborn calves —A field-study with specific egg antibodies [J]. Berl. Munch. Tierarztl. Wochenschr.,1993,106(11):383-387.
[128]Ikemori Y, Kuroki M, Peralta R C, et al. Protection of neonatal calves against fatal enteric colibacillosis by administration of egg-yolk powder from hens immunized with k99-piliated enterotoxigenic Escherichia coli [J]. Am. J. Vet. Res.,1992,53(11):2005-2008.
[129]Kuroki M, Ikemori Y, Yokoyama H, et al. Passive protection against bovine rotavirus-induced diarrhea in murine model by specific immunoglobulins from chicken egg-yolk [J]. Vet. Microbiol., 1993,37(1-2):135-146.
[130]Yokoyama H, Peralta R C, Umeda K, et al. Prevention of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk Salmonella-specific antibodies [J]. Am. J. Vet. Res., 1998,59(4):416-420.
[131]Hirai K., Arimitsu H, Umeda K, et al. Passive Oral Immunization by Egg Yolk Immunoglobulin (IgY) to Vibrio cholerae Effectively Prevents Cholera [J]. Acta Med. Okayama,2010,64(3): 163-170.
[132]LeClaire R D, Hunt R E, Bavari S. Protection against bacterial superantigen staphylococcal enterotoxin B by passive vaccination [J]. Infect. Immun.,2002,70(5):2278-2281.
[133]胡思顺.家畜DNA疫苗免疫最新进展及应用前景[J].《国外医学》预防、诊断、治疗用生物制品分册,2002,25(3)：107-110.
[134]Kirman J R, Turon T, Su H, et al. Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen 85A [J]. Infect. Immun.,2003, 71:575-579.
[135]Deliyannis G, Boyle J S, Brady J L, et al. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge [J]. Proc. Natl. Acad. Sci. U. S. A.,2000,97(12): 6676-6680.
[136]Baldwin S L, D'Souza C D, Orme 1 M, et al. Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A [J]. Tuber. LungDis.,1999,79:251-259.
[137]Leong J, Vinal A C, Dallas W S. Nucleotide-sequence comparison between heat-labile toxin b-subunit cistrons from Escherichia coli of human and porcine origin [J]. Infect. Immun.,1985, 48(1):73-77.
[138]So M, Mccarthy B J. Nucleotide-sequence of the bacterial transposon tn 1681 encoding a heat-stable (st) toxin and its identification in entero-toxigenic Escherichia coli strains [J]. Proc. Natl. Acad. Sci. U. S. A.-Biol. Sci.,1980,77(7):4011-4015.
[139]Lee C H, Moseley S L, Moon H W, et al. Characterization of the gene encoding heat-stable toxin-ii and preliminary molecular epidemiological-studies of enterotoxigenic Escherichia coli heat-stable toxin-II producers [J]. Infect. Immun.,1983,42(1):264-268.
[140]卢圣栋.现代分子生物学实验技术[M].第二版.北京：中国协和医科大学出版社,1999.
[141]汪家政,范明.蛋白质技术手册[M].北京：科学出版社,2000.
[142]Kunkel S L, Robertson D C. Purification and chemical characterization of the heat-labile enterotoxin produced by enterotoxigenic Escherichia coli [J]. Infect. Immun.,1979,25(2): 586-596.
[143]Saeed A M K, Sriranganathan N, Cosand W, et al. Purification and characterization of heat-stable enterotoxin from bovine enterotoxigenic Escherichia coli [J]. Infect. Immun.,1983,40(2): 701-707.
[144]Mundell D H, Anselmo C R, Wishnow R M. Factors influencing heat-labile Escherichia coli enterotoxin activity [J]. Infect. Immun.,1976,14(2):383-388.
[145]Staples S J, Asher S E, Giannella R A. Purification and characterization of heat-stable enterotoxin produced by a strain of E. coli pathogenic for man [J]. J. Biol. Chem.,1980,10:4716-4721.
[146]Akita E M, Nakai S. Immunoglobulins from egg yolk:isolation and purification [J]. J. Food Sci., 1992,57(3):629-634.
[147]Maya D C, Vasantha B M, Vijayan L A, et al. An improved method for isolation of anti-viper venom antibodies from chicken egg yolk [J]. J. Biochem. Biophys. Meth.,2002,51(2):129-138.
[148]Whipp S C. Assay of enterotoxigenic Escherichia coli heat-stable toxin b in rats and mice [J]. Infect. Immun.,1990,58:930-934.
[149]Wu C M, Chung T C. Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein [J]. FEMS Immunol. Med. Microbiol.,2007,50(3):354-65.
[150]李建荣,于涟,黄耀伟,等.免疫刺激复合物(ISCOM)介导的传染性法氏囊病病毒多聚蛋白基因免疫的研究[J].病毒学报,2001,17(4)：341-348.