Efficient immunodiagnosis of Citrus yellow mosaic virus using polyclonal antibodies with an expressed recombinant virion-associated protein
Efficient immunodiagnosis of Citrus yellow mosaic virus using polyclonal antibodies with an expressed recombinant virion-associated protein
复制标题
使用具有表达的重组病毒体相关蛋白的多克隆抗体对柑橘黄花叶病毒进行有效的免疫诊断
作者：
P. V. Kumar;S. K. Sharma;N. Rishi;V. Baranwal
柑橘黄花叶病毒（Citrus yellow mosaic virus，CYMV）是花椰菜病毒科Badnavirus属的一种病毒。它是柑橘黄花叶病的病原，导致产量下降。由于病毒通过嫁接进行无性繁殖，因此开发基于血清学技术的高通量诊断方法是生产健康无病毒种植材料的先决条件。目前的研究描述了多克隆抗体的发展提出了针对纯化的重组病毒体相关蛋白（rVAP）编码的CYMV的ORF-II的兔。用免疫电镜（ISEM）、抗原包被平板-酶联免疫吸附试验（ACP-ELISA）和免疫捕获PCR（IC-PCR）检测抗血清的特异性。该抗血清在1：2000的ACP-ELISA中特异性反应，用于检测CYMV感染的植物。通过ACP-ELISA筛选保持在玻璃房中的CYMV感染的植物来验证抗血清。据我们所知，这是第一个报告，利用重组病毒体相关蛋白作为融合蛋白的多克隆抗血清，可用于筛选CYMV感染植物的ELISA和IC-PCR。这些免疫诊断方法可有效地应用于柑桔的常规指标鉴定和检疫检验。
Citrus yellow mosaic virus (CYMV) is a member of genus Badnavirus of the family Caulimoviridae. It is the causal agent of citrus yellow mosaic disease in citrus and causes reduction in yield. As the virus is vegetative propagated by grafting, development of high-throughput diagnosis methods based on serological techniques is a prerequisite for production of healthy virus-free planting material. The current study describes the development of polyclonal antibodies raised in rabbits against purified recombinant virion-associated protein (rVAP) encoded by ORF-II of CYMV. The specificity of developed antiserum was evaluated in immunosorbent electron microscopy (ISEM), antigen-coated plate-enzyme linked immunosorbent assay (ACP-ELISA) and immunocapture PCR (IC-PCR). The antiserum specifically reacted up to a dilution of 1:2000 in ACP-ELISA for detection of CYMV-infected plants. The antiserum was validated by screening CYMV-infected plants maintained in the glass house through ACP-ELISA. To the best for our knowledge, this is the first report on production of polyclonal antiserum using recombinant virion-associated protein as fusion protein, which could be used for screening CYMV-infected plants by ELISA and IC-PCR. These immunodiagnostic methods can be effectively employed in routine indexing of citrus and in quarantine process.