摘要
利用纯化的重组杆状病毒表达猪圆环病毒2型(PCV2)Cap蛋白作为检测抗原,建立一种间接ELISA方法用于血清抗体检测。昆虫细胞株(Sf-21)接种重组杆状病毒,对蛋白表达参数及纯化工艺进行了优化,制备了5批重组蛋白抗原,表达量占总蛋白的18.7%左右;Ni2+亲和层析柱纯化获得重组蛋白纯度为90.1%;重组蛋白可被特异性抗体识别,具有良好的免疫活性。用建立的ELISA法对30份已知阴性血清样本检测临界OD405nm值为0.343;ELISA与IPMA对150份血清进行平行检测符合率为95.3%,特异性为91.2%,敏感性为96.5%;与其他几种常见猪病毒参考血清无交叉反应。组装的试剂盒批内和批间变异系数分别为4.0%～4.8%和8.9%～9.9%;置-20℃保存12个月稳定。试剂盒对人工感染猪血清检测结果,接种后第5周抗体阳转,第8周～9周抗体水平达到高峰;对来自黑龙江、吉林、河北、上海等地猪场1085份血清抗体检测,阳性率为73.6%。研制的ELISA试剂盒为临床PCV2血清抗体检测及疫苗免疫效果的评价提供了技术手段。
An indirect ELISA was developed for detecting antibodies against porcine circovirus type 2 （PCV2） using baculovirus-expressed recombinant capsid （Cap） protein as antigen. Spodoptera frugiperda （Sf-21） insect cells were inoculated with the recombinant baculovirus and Cap products were identified by Western blot purified under optimized conditions. In comparison with immunoperoxidase monolayer assay （IPMA）, there was 95.3 % coincidence between the ELISA and IPMA based on detection of 150 serum samples..The assay showed no cross-reactivity with the reference sera of other swine virus. The variant coefficient of ELISA test kit in a batch and between batches varied from 4.0 % to 4.8 % and from 8.9 % to 9.9 %, respectively. The ELISA kit was stable at -20 ℃ at least for one year. Antibodies could be detected fi＇om artificial infected swine sera from 5 weeks post inoculation, and reached a peak at 8 to 9 weeks PI. Test of 1085 serum samples collected from Heilongjiang, Jilin, Hebei provinces and Shanghai regions showed 73.6 % positive.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2008年第7期548-551,578,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家"948"计划(2006-Z6)
国家科技支撑计划项目资助(2006BAD06A07)
