摘要
【目的】建立一种检测大熊猫血清中犬细小病毒(canine parvovirus,CPV)抗体的间接酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)方法。【方法】以大熊猫源CPV DNA为模板,利用PCR扩增VP2基因,再用原核表达系统对VP2基因进行表达,经纯化后的蛋白作为ELISA包被抗原;制备并纯化兔抗大熊猫IgG,采用辣根过氧化物酶(horseradish peroxidase,HRP)标记作为间接ELISA酶标二抗,通过棋盘法确定抗原包被质量浓度及血清稀释度等条件,并评估建立方法与商业试剂盒检测样品结果的符合率。【结果】原核表达成功获得约70 ku的VP2蛋白,将其作为间接ELISA包被抗原,抗原最佳包被质量浓度为2.0μg/mL,血清最佳稀释度为1∶400,用该方法进行检测血清样品时OD450≥0.258为阳性,反之为阴性。采用建立的ELISA方法检测大熊猫血清样本阳性率为60.0%,高于商业化检测试剂盒检测的阳性率(52.5%),两者总符合率达到87.5%。【结论】本研究首次建立了基于大熊猫源CPV VP2蛋白为抗原、自制HRP标记兔抗大熊猫IgG为酶标二抗的间接ELISA方法,该方法特异性强、灵敏性较高、重复性好,为大熊猫血清中CPV抗体的检测提供了技术支持。
[Purpose]To establish an indirect enzyme linked immunosorbent assay(ELISA)method for detecting canine parvovirus(CPV)antibodies in giant panda serum.[Methods]The VP2 gene was amplified by PCR using CPV DNA derived from giant pandas as a template,then the VP2 gene was expressed using a prokaryotic expression system,and the purified protein was used as an ELISA coated antigen.Rabbit anti-giant panda IgG was prepared and purified,and labeled with horseradish peroxidase(HRP)as an indirect ELISA enzyme-labeled secondary antibody.The conditions such as the mass concentration of coated antigen and dilution of serum were determined by the checkerboard method;and the consistency rate between the established method and the test sample results of commercial kits was evaluated.[Results]Prokaryotic expression successfully obtained about 70 ku of VP2 protein,which was used as an indirect ELISA coated antigen.The optimal mass concentration of coated antigen was 2.0μg/mL,and the optimal dilution of serum was 1∶400.When using this method to detect serum samples OD450≥0.258 was positive,and vice versa was negative.The positive rate of giant panda serum samples detected by the ELISA method established in this study was 60.0%,which was higher than the positive rate of commercial detection kits(52.5%),and the total coincidence rate reached 87.5%.[Conclusion]This study establishs an indirect ELISA method based on the giant panda-derived CPV VP2 protein as the antigen,and homemade HRPlabeled rabbit anti-giant panda IgG as the enzyme-labeled secondary antibody for the first time.This method has strong specificity,high sensitivity and good repeatability,and provides technical support for the detection of CPV antibodies in giant panda serum.
作者
李强
严立恒
兰景超
邓英
孙珊珊
罗娌
史纪强
颜其贵
LI Qiang;YAN Liheng;LAN Jingchao;DENG Ying;SUN Shanshan;LUO Li;SHI Jiqiang;YAN Qigui(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Chengdu Research Base of Giant Panda Breeding,Chengdu 610081,China)
出处
《云南农业大学学报(自然科学版)》
CSCD
北大核心
2023年第5期795-802,共8页
Journal of Yunnan Agricultural University:Natural Science
基金
成都大熊猫繁育基金会项目(CPF2017-35)
成都大熊猫繁育研究基地自立课题(2021CPB-C13)。