鸽圆环病毒抗体间接ELISA检测方法的建立及应用
Establishment and application of indirect ELISA method for the detection of pigeon circovirus antibodies
杨颖 1路文彬 1向凤君 1雷白时 1赵款 1安峥 2张武超 1袁万哲1
作者信息
- 1. 河北农业大学动物医学院,河北保定 071001
- 2. 保定市久盟生物科技有限公司,河北保定 071001
- 折叠
摘要
为建立一种检测鸽圆环病毒(pigeon circovirus,PiCV)抗体的间接ELISA方法,本研究将大小为828 bp的PiCV Cap基因片段克隆至载体pGEX-4T-1,通过低温诱导获得了可溶性Cap重组蛋白,经Western-blot验证后发现,该重组蛋白与抗GST标签抗体和抗Cap蛋白抗体均可发生特异性反应.利用纯化的Cap重组蛋白作为包被抗原,通过棋盘滴定法优化各反应条件,建立了PiCV抗体间接ELISA检测方法.结果显示,最佳抗原包被浓度为0.25mg/L,5％BSA作为封闭液37 ℃孵育2 h,待检血清最佳稀释度为1∶800,孵育时间为45 min,家兔抗鸽IgG-HRP二抗最佳稀释度为1∶8000,孵育时间为30 min,最佳显色时间为15 min,当检测样品D450≥0.341则判定其为阳性.用该方法检测鸽腺病毒、鸽疱疹病毒、鸽Ⅰ型副黏病毒和轮状病毒的阳性血清时结果呈阴性,对稀释至1∶12 800的阳性血清进行检测时结果仍为阳性,批内和批间变异系数均小于10％.与常规PCR符合率为88.2％.用该方法对86份临床血清样品进行检测,阳性率为17.4％.综上所述,该间接ELISA方法的建立,作为一种特异性强、灵敏度高、重复性好的血清学检测方法,为后续PiCV候选疫苗免疫后抗体水平的评价及PiCV感染状况的评估提供了技术支持.
Abstract
In order to establish an indirect ELISA method for detection of pigeon circovirus(PiCV)antibodies,PiCV Cap gene fragment with a size of 828 bp was cloned into pGEX-4T1 vector,and the solu-ble Cap recombinant protein was obtained by low temperature induction,which was verified by Western-blot and found to react specifically with both anti-GST tag antibodies and anti-Cap protein antibodies.The purified Cap recombinant protein was used as coating antigen,an indirect ELISA method for detec-tion of PiCV antibody was established by optimizing each reaction condition through checkerboard titration method.The results showed that the optimal antigen coating concentration was 0.25 mg/L,5％BSA as a blocking solution,incubation at 37 ℃ for 2 h,the optimal dilution of the serum to be tested was 1∶800,the incubation time was 45 min,the optimal dilution of rabbit anti-pigeon IgG-HRP secondary an-tibody was 1∶8 000,the incubation time was 30 min,and the optimal color development time was 15 min.When the serum sample D450≥0.341,it was judged as positive.The positive serological results of pigeon adenovirus,pigeon herpesvirus,pigeon paramyxovirus type Ⅰ and rotavirus were negative,and the posi-tive serum diluted to 1∶12 800 could still be detected,with intra-assay and inter-assay coefficients of variation of less than 10％.The coincidence rate of indirect ELISA method with conventional PCR method was 88.2％.The indirect ELISA method was used to detect 86 clinical serum samples,and the posi-tive rate was 17.4％.The indirect ELISA method is highly specific,sensitive and repeatable,which pro-vides technical support for the detection of antibody levels after PiCV vaccine candidate immunization andPiCV epidemiological investigation.
关键词
鸽圆环病毒/Cap蛋白/原核表达/间接ELISA方法Key words
pigeon circovirus/Cap protein/prokaryotic expression/indirect ELISA引用本文复制引用
基金项目
河北省"三三三人才工程"项目(A20221003)
校企合作项目(3109120001)
出版年
2024
国家科技期刊平台
NSTL
万方数据