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

从 RF-dsRNA 分子克隆植物病毒基因并生产针对该基因编码的蛋白质的抗血清。

基本信息

批准号：
07660050
负责人：
NATSUAKI Tomohide
金额：
$ 1.47万
依托单位：
Ustunomiya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660050/
关键词：
Plant virus RF-dsRNA Cloning Sequencing unpurifable virus シークエンス融合タンパク質 RT-PCR

项目摘要

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.

植物病毒的分子克隆已在过去十年中进行。克隆植物病毒的目标之一是改进病毒检测和诊断。作为 cDNA 合成的模板，通常从纯化的病毒制剂中以相对纯净的形式和相当大量地提取 RNA 或 DNA。这些策略依赖于从受感染植物中纯化病毒颗粒。然而，有许多顽固病毒或病毒分离株无法通过现有方法纯化，因此无法使用标准核酸模板进行克隆。对于没有可用抗血清的病毒，需要替代的检测和诊断方法。对于其中几种病毒，应用从草本或木本宿主中提取 dsRNA 技术可以检测病毒复制核酸 (RF-dsRNA)。本研究的目的是利用从病毒感染植物中纯化的 dsRNA 生成 cDNA 克隆。以病毒感染组织中提取的dsRNA为模板，进行cDNA合成和PCR，完成了柑橘tristeza病毒的分子克隆。该方法对于无法获得纯化病毒制剂的其他植物病毒应该具有通用性。此外，将通过PCR扩增的cDNA与表达载体中的蛋白A基因融合，并获得融合蛋白来免疫兔子。所得的抗血清与CTV感染的植物中表达的非结构蛋白反应。