摘要
猪繁殖与呼吸综合征病毒（PRRSV）是影响世界养猪业的一种重要病原，GP5蛋白是病毒编码的重要免疫保护蛋白。目前，虽然PRRSV抗体检测方法较多。但尚无商品化的GP5蛋白ELISA抗体检测试剂盒。本研究根据高致病性PRRSVBB0907毒株GP5基因特性．删除了GP5基因跨膜区，并将剩余基因片段优化为大肠杆菌偏嗜性，构建了高效表达的GP5重组蛋白的原核表达载体．制备获得GP5重组蛋白。通过反应条件优化，建立了PRRSVGP5蛋白间接ELISA（GP5-ELISA）抗体检测方法，其最佳抗原包被浓度为1．0μg／mL，血清的最佳稀释度为1：100，与猪圆环病毒2型（PCV2）、猪瘟病毒（CSFV）、伪狂犬病病毒（PRV）、口蹄疫病毒（FMDV）、脑心肌炎病毒（EMCV）和副猪嗜血杆菌（HPS）等病原抗体均无交叉反应。相对于IDEXX公司PRRSVELISA抗体检测试剂盒，本研究建立的间接GP5-ELISA的敏感性和特异性分别为90．69％和92.86％,两种方法的符合率为91．11％。用本研究建立的间接GP5-ELISA对360份临床猪血清的PRRSV抗体进行检测，抗体阳性率为74．44％。上述结果表明，本研究建立的猪繁殖与呼吸综合征病毒GP5蛋白间接ELISA抗体检测方法具有广阔的应用前景。
Porcine reproductive and respiratory syndrome virus（PRRSV）is an important pathogen affecting the world pig industry. The GP5 protein is an important immunoprotective protein encoded by the virus. At present,there are a lot methods developed for detection of anti-PRRSV antibodies,but no commercially ELISA kit is available for the detection of GP5 protein antibodies specially. This study was based on the GP5 gene characteristics of a highly pathogenic PRRSV BB0907 strain,deleting the transmembrane region of the GP5 gene,and the remaining gene fragments were optimized for E. coli biophagy. A prokaryotic expression vector was constructed for the highly expressing recombinant protein of GP5,and the modified GP5 recombinant protein was expressed and purified. And an indirect ELISA method was established by optimizing the reaction conditions. The optimum antigen concentration was 1. 0 μg/mL,the optimal dilution of serum was 1：100. It had no cross reaction with the antibodies against porcine circovirus type 2,classical swine fever virus,pseudorabies virus,foot-and-mouth disease virus,encephalomyocarditis virus and parssuis haemophilus,respectively. Compared with the commercial PRRSV ELISA kit,it had the relative sensitivity of 90.69%and specificity of 92.86%. The coincidence rate of the two methods was 91.11%. The positive rate of PRRSV antibodies in 360 clinical pig serum was 74. 44%. In conclusion,the developed indirect ELISA for the detection antibodies against GP5 protein of porcine reproductive and respiratory syndrome virus in this study has an important application prospect.
作者
侯贺飞
刘雪威
白娟
宋中宝
姜平
HOU He-fei;LIU Xue-wei;BAI Juan;SONG Zhong-bao;JIANG Ping(Key Lab oratory of Animal Bacteriology,Ministry of Agri ulture/College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2018年第9期1086-1092,共7页
Chinese Veterinary Science
基金
国家自然科学基金项目(31672565)
国家生猪产业技术体系专项(CARS-36)
