猪源ETEC菌毛基因的多重PCR检测及其菌毛抗体的研制
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摘要
初生和断奶仔猪大肠杆菌性腹泻病的主要病原是肠毒素性大肠杆菌(ETEC)。ETEC的致病作用与其具有粘附性菌毛和产肠毒素密切相关，二者缺一不可。ETEC致病过程中重要的一个环节是细菌借粘附性菌毛粘附固着在仔猪小肠绒毛上皮细胞上，继而增殖并产生大量肠毒素引起小肠吸收分泌功能失常而致腹泻。本文针对粘附性菌毛这一环节，采用热抽提法提取四种大肠杆菌粘附性菌毛，免疫产蛋母鸡，获得高效价抗菌毛卵黄抗体。免疫后，四种菌毛都能诱导鸡产生卵黄抗体，其中K88菌毛免疫性最好，诱导抗体效价高而且能长时间维持，987p菌毛能快速诱导卵黄抗体的产生，但整体效价低。不同佐剂制备的K83菌毛蛋白苗的免疫结果表明铝胶佐剂能快速诱导抗体的产生，蜂胶佐剂苗诱导的抗本持续时间短，弗氏佐剂苗和白油佐剂苗能诱导高效价抗体的产生并能长时间维持。用获得的卵黄抗体进行仔猪小肠上皮细胞粘附和粘附抑制试验表明卵黄抗体能显著且特异地抑制相应菌毛菌对小肠上皮细胞的粘附。对攻毒仔猪进行口服卵黄抗体治疗，试验结果表明卵黄抗体可减轻仔猪腹泻程度和缩短仟猪排毒时间及减少排毒量。肠毒素性大肠杆菌有两类毒力因子，肠毒素和粘附性菌毛．检测肠毒素性大肠杆菌一般也是针对这两类毒力因子进行的。本实验根据致仔猪腹泻常见的四种粘附性菌毛的结构基因保守区段序列，设计合成了四对引物，经聚合酶链式反应可分别扩增出201bp的K88基因片段、314bp的K99基因片段、380bp的F41基因片段、459bp的987p基因片段。各扩增产物长度和限制性酶酶切结果表明均为预期产物。用这四对引物组成一个检测四种菌毛基因的多重PCR系统，对各种标准菌毛阳性菌株及其不同组合和各种阴性对照菌株的检测结果表明此多重PCR系统特异性好，敏感性较高，可用于四种菌毛基因的检测。
Enterotoxigenic Escherichia Coli (ETEC) is the main pathogen of
colibacillosis of neonatal and post weaned piglets. The pathogenesis of ETEC is strongly related to adhesive fimbriae and enterotoxin. Mechanisms in the pathogenesis of piglets diarrhea caused by ETEC is that ETEC attach to the small intestine by means of the adhesive fimbriae and replicate and elaborate enterotoxin that interfere the biochemistry of small intestine cell. In this experiment, four kinds of Enterotoxigenic Escherichia coli fimbriae were extracted by thermoextract method. For K99,F41 and 987p fimbriae , vaccines were made from each fimbriae and Freund's adjuvant. For K88 fimbriae, vaccines were made by Freund's adjuvant, white oil adjuvant, A1(OH)3 adjuvant and propolis adjuvant respectively. And vaccine containing four fimbriae was made by Freund's adjuvant. Laying chickens were immunized with different vaccines. The result indicated that all four fimbriae could cause the immunity reaction. K88 fimbriae could induce the highest level egg yolk antibodies which could sustain a long time. And the
Freund's adjuvant and the White oil adjuvant vaccine could induce the highest level egg yolk antibodies and could keep stable within a long time. Trial of adhesion in vitro indicated that the attachment of bacterial cells to intestinal epithelial cells was strongly inhibited when homologous anti-fimbrial antibody solutions were used. The anti-K88 antibody reduced considerably the severity of diarrhea in piglets challenged with K88+ETEC. All results indicate that the chicken egg yolk antibody against ETEC fimbriae was effective. For detection, adhesive fimbriae and enterotoxin are the markers of ETEC. To detect four kinds of adhesive fimbriae genes of ETEC from porcine, four pairs of primers are designed and synthesized according to the conserve sequence region of four kinds of fimbriae structural gene. Four pairs of primers could amplify 201bp,314bp,380bp and 459bp segment respectively. The length and the results of restriction enzyme digestion indicate that the amplified products are respected. A multiplex
polymerase chain reaction(PCR) system for the rapid screening of K88,K99,F41 and 987p genes was developed. The specificity of this
multi-PCR system was confirmed by PCR of ETEC reference strains and negative control bacterial strains. Results also show that the multi-PCR was sensitive. It will be useful for identification of K88, K99JF41 and 987p genes and diagnosis of procine colibacillosis.
Enterotoxigenic Escherichia Coli (ETEC) is the main pathogen of
colibacillosis of neonatal and post weaned piglets. The pathogenesis of ETEC is strongly related to adhesive fimbriae and enterotoxin. Mechanisms in the pathogenesis of piglets diarrhea caused by ETEC is that ETEC attach to the small intestine by means of the adhesive fimbriae and replicate and elaborate enterotoxin that interfere the biochemistry of small intestine cell. In this experiment, four kinds of Enterotoxigenic Escherichia coli fimbriae were extracted by thermoextract method. For K99,F41 and 987p fimbriae , vaccines were made from each fimbriae and Freund's adjuvant. For K88 fimbriae, vaccines were made by Freund's adjuvant, white oil adjuvant, A1(OH)3 adjuvant and propolis adjuvant respectively. And vaccine containing four fimbriae was made by Freund's adjuvant. Laying chickens were immunized with different vaccines. The result indicated that all four fimbriae could cause the immunity reaction. K88 fimbriae could induce the highest level egg yolk antibodies which could sustain a long time. And the
Freund's adjuvant and the White oil adjuvant vaccine could induce the highest level egg yolk antibodies and could keep stable within a long time. Trial of adhesion in vitro indicated that the attachment of bacterial cells to intestinal epithelial cells was strongly inhibited when homologous anti-fimbrial antibody solutions were used. The anti-K88 antibody reduced considerably the severity of diarrhea in piglets challenged with K88+ETEC. All results indicate that the chicken egg yolk antibody against ETEC fimbriae was effective. For detection, adhesive fimbriae and enterotoxin are the markers of ETEC. To detect four kinds of adhesive fimbriae genes of ETEC from porcine, four pairs of primers are designed and synthesized according to the conserve sequence region of four kinds of fimbriae structural gene. Four pairs of primers could amplify 201bp,314bp,380bp and 459bp segment respectively. The length and the results of restriction enzyme digestion indicate that the amplified products are respected. A multiplex
polymerase chain reaction(PCR) system for the rapid screening of K88,K99,F41 and 987p genes was developed. The specificity of this
multi-PCR system was confirmed by PCR of ETEC reference strains and negative control bacterial strains. Results also show that the multi-PCR was sensitive. It will be useful for identification of K88, K99JF41 and 987p genes and diagnosis of procine colibacillosis.
引文
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2、Kohler. E. M.. Studies on Escherichia Coli in gnotobiotic pigs. Can. J. Comp. Med. 1967, 31: 278-283
3、Smith H. W. et al.. Studies in Escherfchia Coli enterotoxins. J. Pathol. Bacteriol. 1967, 93: 531
4、Levine M M. Escherfchia Coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhaic and enteroadherent. J. Inf. Dis. 1987, 155: 377-389
5、Osek J. et al. Occurrence of fimbriae and enterotoxins in E. Coli strains isolated from piglets in Poland. Comparative Immunology Microbiology and Infectious Diseases, 1992, 15(4): 285-292
6、Wilson M R. Enteric collibacillosis in: diseases of swine. The Iowa Stater University Press, 1986
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