Molecular cloning of plant virus gene from RF-dsRNA and the production of antisera against proteins encoded by the gene.

植物病毒的分子克隆在过去的十年中已经完成。克隆植物病毒的目的之一是提高病毒的检测和诊断水平。作为cDNA合成的模板，RNA或DNA通常以相对纯的形式和相当大量地从纯化的病毒制备物中提取。这些策略依赖于从受感染的植物中纯化病毒颗粒。然而，有许多寄生病毒或病毒分离物不能用现有的方法纯化，因此，标准核酸模板不能用于它们的克隆。对于没有可用抗血清的病毒，需要替代的检测和诊断方法。对于这些病毒中的几种，从草本或木本宿主中应用dsRNA提取技术已经允许检测病毒复制核酸（RF-dsRNA）。本研究的目的是从病毒感染的植物中纯化的dsRNA产生的cDNA克隆的生产。以柑橘衰退病毒（Citrus tristeza virus，TRV）侵染组织中提取的dsRNA为模板，通过cDNA合成和PCR扩增，完成了柑橘衰退病毒（Citrus tristeza virus，TRV）的分子克隆。该方法对于不能获得纯化病毒制剂的其他植物病毒具有普遍实用性。将PCR扩增的cDNA与Protein A基因融合，构建表达载体，获得融合蛋白，免疫家兔。所得抗血清与CTV感染植物中表达的非结构蛋白反应。

Molecular cloning of plant viruses has been carried out during the past decade. One objective of cloning plant viruses has been the improvement of virus detection and diagnosis. As templates for cDNA synthesis, RNA or DNA are usually extracted from purified virus preparations in relatively pure form and in rather large amounts. These strategies rely on the purification of virus particles from infected plants. However, There are many recalcitrant viruses or virus isolates that can not be purified by current methods and, therefore, the standard nucleic acid templates are not accessible for their cloning. It is the viruses for which there are no available antisera that alternate methods of detection and diagnosis are needed. For several of these viruses, the application of dsRNA extraction techniques from herbaceous or woody hosts has permitted the detection of virus replicative nucleic acids (RF-dsRNA). The objective of this study were the production of cDNA clones generated from dsRNA purified from virus-infected plants. The molecular cloning of citrus tristeza virus by using dsRNA that were extracted from virus-infected tissue as the template for cDNA synthesis and PCR was sccomplished. The method should have general utility for other plant viruses where purified virus preparations can not be obtained. Furthermore, the cDNA amplified by PCR was fused to the Protein A gene in an expression vector and the fusion protein was obtained to immunize a rabbit.. The resulting antiserum reacted with non-structural protein expressed in CTV-infected plants.