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鸭疫里默氏杆菌重组蛋白P25间接ELISA
检测方法的建立
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辜新贵1，2,袁建丰2，3 ，覃宗华2 ，于三科1，谢明权2,吕敏娜2，余劲术2，
吴彩艳2 ，蔡建平2*
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（1.西北农林科技大学 动物医学院，陕西 杨凌712100；2.广东省农业科学院 兽医研究所
广东省兽医公共卫生公共实验室，广东 广州510640； 3.华南农业大学 兽医学院，广东 广州510642）
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摘要：
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以鸭疫里默氏杆菌血清1型(RA 1)基因组文库中筛选获得的一种“保守假定蛋白P25”基因的重组表达产物为包被抗原，建立了检测RA血清抗体的间接ELISA。诱导表达的重组P25蛋白纯化后进行包被，十字交叉滴定试验确定，此ELISA的最适抗原包被浓度为0.6μg/mL，血清最佳稀释度为1∶64，与大肠杆菌、多杀性巴氏杆菌感染鸭血清无交叉反应。以RA 1的P25为包被抗原对RA 2、5、8、10血清型的标准分型血清和经临床剖检诊断的病鸭血清进行检测，其结果均为阳性。以RA 1型的P25基因设计的特异性引物分别扩增出了RA 2、5、8、10的P25基因。所扩增的P25基因的核苷酸序列及其编码的氨基酸序列和抗原表位在这些不同血清型间高度保守，提示此P25蛋白是各种血清型RA间的共同抗原，所建立的ELISA可以用于检测不同血清型RA的感染。 |
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关键词：
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鸭疫里默氏杆菌；重组P25蛋白；间接ELISA；共同抗原 |
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中图分类号：S 855.12
文献标识码：
A 文章编号：1673-4696(2008)03-0218-06 |
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Development of an indirect ELISA using recombinant P25 protein
for detecting Riemerella anatipestifer infection in ducks |
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GU Xin-gui1,2,YUAN Jian-feng2,3,QIN
Zong-hua2 ,YU San-ke1,XIE Ming-quan2，
L Min-na2,YU Jin-shu2,WU Cai-yan2,CAI
Jian-ping2 |
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(1.College of Veterinary Medicine,Northwest Sci-Tech University of Agriculture and Forestry,Yangling 712100,China;
2.Public Laboratory of Veterinary Public Health of Guangdong Province/Institute of Veterinary Medicine,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;
3.College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China) |
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Abstract: An indirect ELISA was developed for detecting serum antibody against Riemerella anatipestifer(RA) in ducks,using the recombinant expression product of the gene encoding “conserved hypothe-tical protein P25” as the coating antigen.The gene was identified by screening RA 1 genomic library using the antisera against the capsule and outer membrane proteins of RA 1.The ORF encoding P25 was recombined into Escherichia coli Rosetta and expressed with an induction of 1mmol/L IPTG at 37℃.The purified recombinant P25 was coated on the ELISA plate for detecting the specific antibodies against RA.The optimal conditions for coating antigen concentration and antisera dilution were 0.6μg/mL and 1∶64，respectively.This indirect ELISA could be applied to probe the different serotypes of RA infection which was verified experimentally by the assay of the specific antisera against RA 1,2,5,8 and 10 serotype.It was further demonstrated that P25 was a common antigen among serotypes of RA.These results showed that this indirect ELISA could be used for detecting duck infections with different serotypes of R.anatipestifer. |
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Key words: Riemerella anatipestifer;recombinant P25 protein;indirect ELISA;common antigen |
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