摘要
本研究旨在建立一种检测猪嵴病毒(PKV)抗体的间接ELISA检测方法。通过原核表达系统表达PKV VP0基因作为包被抗原,并通过Western blot鉴定证明重组蛋白与PKV阳性血清能发生良好的特异性反应。优化反应条件,得出PKV VP0间接ELISA检测方法最适反应条件是抗原包被浓度为0.62μg/mL,血清稀释浓度为1∶200,最佳酶标二抗稀释浓度1∶5000,使用5%脱脂奶粉作为封闭液37℃孵育1 h,显色10 min,当检测样品OD_(450)<0.225判定血清样品抗体为阴性,OD_(450)≥0.225判定血清样品抗体为阳性,且特异性高、重复性好,与Western blot符合率达到95.6%。PKV VP0蛋白间接ELISA方法的建立,作为一种操作简单,灵敏度高,特异性高,重复性好的血清学检测方法,为PKV的监测和预防提供了技术支持。
This study aimed to develop an indirect ELISA method for detecting Porcine kobuvirus (PKV) antibodies.The PKV VP0 gene was expressed as a coating antigen through a prokaryotic expression system.The resulting VP0 protein was confirmed in Western blot using PKV positive serum.The indirect ELISA method was optimized as the antigen coating concentration at 0.62μg/mL,serum dilution concentration at 1∶200,enzyme-labeled secondary antibody dilution at 1∶5000,5% skimmed milk powder as a blocking solution,incubation at 37°C for 1 hour and color development for 10 minutes.When the test sample OD_(450)<0.225,the serum sample was determined to be negative while OD_(450)≥0.225 to be positive.The indirect ELISA method developed using recombinant PKV VP0 protein had the advantages such as simple operation,and high sensitivity,specificity and repeatability.Therefore,this method could be used for the monitoring and prevention of PKV.
作者
韩磊
彭志豪
刘莹
顾文源
王丙雷
郭禹
左玉柱
范京惠
HAN Lei;PENG Zhihao;LIU Ying;GU Wenyuan;WANG Binglei;GUO Yu;ZUO Yuzhu;FAN Jinghui(College of Veterinary Medicine,Hebei Agricultural University,Baoding 07001,China;Animal Diseases Control Center of Hebei,Shijiazhuang 050053,China;Hebei Veterinary Biotechnology Innovation Center,Hebei Agricultural University,Baoding 071001,China)
出处
《中国动物传染病学报》
CAS
北大核心
2023年第1期72-77,共6页
Chinese Journal of Animal Infectious Diseases
基金
河北省重点研发计划项目(19226622D)
河北省农业产业技术体系生猪创新团队(HBCT2018110207)
作者简介
韩磊,男,硕士研究生,预防兽医学专业;通信作者:左玉柱,E-mail:zuoyuzhu@163.com;通信作者:范京惠,E-mail:jinghui76@163.com
