马铃薯Y病毒衣壳蛋白抗原表位分析及其快速ELISA检测方法的建立

Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein

【目的】马铃薯Y病毒(potato virus Y,PVY)是影响马铃薯产量和品质的主要病毒之一,目前尚未发现有效的防治药剂,脱毒种薯的应用是预防PVY危害的主要防治措施。建立灵敏度高、特异性强的PVY快速检测方法,为脱毒种薯质量控制提供技术支撑。【方法】将PVY衣壳蛋白(coat protein,CP)分段表达为有重叠部分的小段多肽,以其为抗原通过Western blot分析PVY-CP的抗原表位。以P/N值最大为标准,通过常规双抗夹心ELISA(DAS-ELISA)对识别不同抗原表位的单克隆抗体(monoclonal antibody,MAb)进行配对试验,筛选一组检测效果最优的配对单克隆抗体,并确认捕获抗体和检测抗体。通过方阵滴定法确定捕获抗体及检测抗体的最佳工作浓度,通过控制变量法确定检测抗体与抗原的最佳共同孵育时间。以不同浓度的PVY-CP蛋白为抗原,检验快速DAS-ELISA的灵敏度。以感染不同病毒的马铃薯样品为抗原,检测快速DAS-ELISA的特异性。同时通过快速DAS-ELISA与RT-PCR检测50份田间采集的疑似感染PVY的马铃薯样品,将二者结果相比较检验检测方法的符合率。运用建立的快速DAS-ELISA对不同株系PVY感染的马铃薯样品进行检测。【结果】利用PVY-CP的6条分段表达多肽筛选到一组能进行DAS-ELISA的配对单克隆抗体(9G6和3D3),并以这对单抗为基础建立了PVY快速DAS-ELISA检测方法。以9G6为捕获抗体包被酶标板,检测抗体3D3经辣根过氧化物酶(horseradish peroxidase,HRP)标记后以2μg·mL^(-1)的工作浓度与抗原在37℃共同孵育5 min。该检测方法检测限为0.5 ng·mL^(-1),特异性分析结果显示该法仅在检测感染PVY的马铃薯样品时呈阳性反应,检测马铃薯S病毒(potato virus S,PVS)、马铃薯M病毒(potato virus M,PVM)、马铃薯卷叶病毒(potato leaf-roll virus,PLRV)等其他常见马铃薯病毒样品均呈阴性反应。通过快速DAS-ELISA与RT-PCR同时对50份田间采集的马铃薯样品进行检测,有48份样品检测结果一致,符合率达96%,且对PVYN及PVYO样品检测结果均呈阳性。【结论】建立的PVY检测方法灵敏度高、特异性强,30 min即可完成检测,方便快捷,为PVY的高通量检测、脱毒种薯的生产提供了关键技术支持。

【Objective】Potato virus Y(PVY) is one of the most serious viruses that affect the yield and quality of potato. At present, no effective virus treatment agent has been found, and application of virus-free seed potato is the main control measure to prevent PVY damage. The objective of this study is to establish a rapid detection method with high sensitivity and specificity, and to provide technical support for quality control of virus-free seed potato.【Method】PVY coat protein(CP) was expressed as small polypeptides with overlapping parts, and the epitopes of PVY-CP were analyzed by Western blot. Matching experiments were performed on monoclonal antibodies that recognized different epitopes by conventional double antibody sandwich ELISA(DAS-ELISA), and a pair of monoclonal antibodies with the best detection effect was screened. At the same time, the captured and detected antibodies were confirmed. According to the standard of maximum P/N value, the working concentration of antibody was determined by square titration. The optimal co-incubation time of antibody and antigen was determined by control variable method. The sensitivity of rapid DAS-ELISA was tested with different concentrations of PVY-CP protein as antigen. Potato samples infected with different viruses were used as antigens to detect the specificity of rapid DAS-ELISA. Through rapid DAS-ELISA and RT-PCR detection of 50 samples of potato samples with suspected infection PVY collected in the field, the coincidence rate was tested. Finally, the established rapid DAS-ELISA was used to detect potato samples infected by different PVY strains.【Result】A panel of monoclonal antibodies(9G6 and 3D3) that can be used for DAS-ELISA were screened using the expressed peptides of PVY CP, and a rapid DAS-ELISA was established based on this panel of MAbs. The ELISA plate coated with the capture antibody 9G6, and then co-incubated with detector antibody HRP labeled 3D3(2 μg·mL^(-1)) and samples for 5 min at 37℃ after blocking the plate. The detection limit was 0.5 ng·mL^(-1). The results of specific analysis showed that the established method was only positive for potato samples infected with PVY, and negative for potato virus S(PVS), potato virus M(PVM) and potato leaf-roll virus(PLRV). In comparison with RT-PCR, DAS-ELSIA had the coincidence rate of 96% through testing 50 potato simples, and the results of PVYN and PVYO potato samples were positive. 【Conclusion】 The established PVY detection method has high sensitivity and specificity, and can be completed within 30 min, which is convenient and fast, and provides key technical support for high-throughput detection of PVY and the production of virus-free seed potato.