摘要
小反刍兽疫病毒(PPRV)的N蛋白是血液中此病毒抗体的主要靶蛋白,本研究克隆并表达N蛋白,将纯化后的N蛋白免疫新西兰大白兔来制备多克隆抗体,采用饱和硫酸铵沉淀法收集多克隆抗体,再用辣根过氧化物酶标记此抗体。用纯化的N蛋白包被96孔板,酶结合物多克隆抗体作为检测抗体,建立了检测小反刍兽疫病毒双抗体阻断ELISA方法,经过反复优化,N蛋白的最佳包被浓度为3.0μg/m L,酶结合物抗体的最佳稀释比例为1∶800,利用本研究确定的方法对200份血清样本进行检测,符合率为95%(169/200),本研究确定的方法可用于检测血清中PPRV的抗体水平,为今后小反刍兽疫的防控和疫苗效果的检测提供基础。
The N protein of Peste des petits ruminants virus(PPRV)is the major target protein in the blood of relevant animals.The gene coding for N protein was cloned for expression in the present study.Then the expressed N protein was purified and used to immunize rabbits.The specific polyclonal antibodies(PAbs)were purified using saturated ammonium sulfate precipitation method and then labeled with horseradish peroxidase(HRP).Development of a double antibody blocking ELISA method was carried out by using the purified N protein as coating antigen for 96-well plates and PAb-HRP conjugate as detector.The optimal parameters included the coating antigen concentration at 3.0μg/mL and PAb-HRP conjugate dilution at 1:800.Total 200 serum samples were tested and 95%(169/200)agreement was obtained.In conclusion,this ELISA method developed in this study could be utilized for detecting PPRV antibodies in serum samples,which would be useful in prevention,control and vaccine efficiency studies.
作者
袁雪涛
王芳蕊
石瑜
杨爱华
YUAN Xuetao;WANG Fangrui;SHI Yu;YANG Aihua(Tianjin Animal Disease Prevention and Control Center,Tianjin 301700,China)
出处
《中国动物传染病学报》
CAS
北大核心
2023年第1期61-65,共5页
Chinese Journal of Animal Infectious Diseases
基金
天津市农业发展服务中心青年科技创新项目(Zxkj202104)
