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十年中得到了广泛的应用。植物病毒克隆的目的之一是提高病毒的检测和诊断能力。作为cDNA合成的模板，RNA或DNA通常是从纯化的病毒制剂中提取的，其形式相对纯净且数量相当大。这些策略依赖于从受感染植物中纯化病毒颗粒。然而，有许多顽固性病毒或病毒分离物无法用现有方法纯化，因此无法获得用于克隆的标准核酸模板。对于没有可用的抗血清的病毒，需要其他检测和诊断方法。对于其中的一些病毒，应用从草本或木质寄主中提取dsRNA的技术可以检测到病毒复制核酸（RF-dsRNA）。本研究的目的是从病毒感染的植物中纯化的dsRNA产生cDNA克隆。利用病毒感染组织中提取的dsRNA作为模板，完成了柑橘tristeza病毒cDNA合成和PCR的分子克隆。对于无法获得纯化病毒制剂的其他植物病毒，该方法应具有普遍效用。将PCR扩增的cDNA与蛋白A基因在表达载体中融合，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发生反应。

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 accomplished. 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.