Zhang G, Leung C, Murdin L, Rovinski B, White K A
Department of Biology, York University, Toronto, Ontario, Canada.
Mol Biotechnol. 2000 Feb;14(2):99-107. doi: 10.1385/mb:14:2:99.
Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.
植物病毒作为在植物中生产外源蛋白（如抗原）的表达载体具有巨大潜力。HIV-1 p24核衣壳蛋白是HIV感染的重要早期标志物，已被用作HIV疫苗开发中的抗原。为了开发一种基于植物的p24生产表达系统，我们研究了使用一种（正）链RNA植物病毒——番茄丛矮病毒（TBSV）作为表达载体。将HIV p24开放阅读框（ORF）作为与TBSV外壳蛋白ORF 5'末端部分的读框内融合体引入到TBSV基因组的克隆cDNA拷贝中。当将对应于工程化病毒载体的体外转录RNA接种到植物原生质体中时具有感染性；Northern和Western印迹分析证实了预测的编码p24的病毒亚基因组mRNA的积累以及p24融合产物的产生。用病毒载体进行的全株感染导致接种叶片中p24融合蛋白的积累，该蛋白与p24特异性抗体发生交叉反应，从而证实了关键抗原决定簇的保留。本研究首次证明TBSV可被工程改造以表达具有临床重要性的完整外源蛋白。文中还讨论了从该病毒载体优化蛋白产量的策略。
