The Food and Environment Research Agency Fera, Sand Hutton, York YO41 1LZ, UK.
J Virol Methods. 2010 Feb;163(2):222-8. doi: 10.1016/j.jviromet.2009.09.024. Epub 2009 Oct 9.
The emerging importance of criniviruses, together with their limited characterisation, necessitates the development of simple tools to enable rapid detection and monitoring in case of an outbreak. While serological tools would be ideal, criniviruses are notoriously difficult to purify and traditional methods of antibody production, requiring purified virus particles, are extremely challenging. The development of a novel molecular strategy for in planta viral antigen preparation to bypass particle purification and allow antibody production are described. An A. tumefaciens-mediated transient expression system, coupled with a green fluorescent protein (GFP) purification method was employed to generate CYSDV coat protein (CP) in whole plant leaves. The CYSDV CP gene was ligated into a GFP construct, transformed into A. tumefaciens and agroinfiltrated into N. benthamiana. Expression levels of the recombinant protein were increased by co-infiltration with the viral gene-silencing suppressor P19 from TBSV. The recombinant protein, purified from plant leaves was used to immunise rats for the preparation of polyclonal antisera.
新兴的弹状病毒的重要性，以及它们有限的特性，需要开发简单的工具来实现快速检测和监测，以防爆发。虽然血清学工具是理想的，但弹状病毒很难纯化，而传统的抗体生产方法，需要纯化的病毒颗粒，极具挑战性。本文描述了一种新型的分子策略，用于在植物体内制备病毒抗原，以绕过颗粒纯化并允许抗体生产。采用根癌农杆菌介导的瞬时表达系统，结合绿色荧光蛋白（GFP）纯化方法，在整株叶片中生成 CYSDV 外壳蛋白（CP）。将 CYSDV CP 基因连接到 GFP 构建体中，转化为根癌农杆菌，并将其农杆菌浸润到 N. benthamiana 中。通过与 TBSV 的病毒基因沉默抑制剂 P19 共浸润，提高了重组蛋白的表达水平。从植物叶片中纯化的重组蛋白被用于免疫大鼠，以制备多克隆抗血清。
