摘要
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。
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 hypothetical 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 recom- bined into Escherichia coli Rosetta and expressed with an induction of 1 mmol/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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第3期218-223,共6页
Chinese Veterinary Science
基金
广东省科技攻关计划项目(2005A20401004)
