Simultaneous production of two foreign proteins from a potyvirus-based vector
Simultaneous production of two foreign proteins from a potyvirus-based vector
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DOI：
10.1016/j.virusres.2005.03.001
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发表时间：
2005-09-01
期刊：
影响因子：
5
通讯作者：
Laliberté, JF
中科院分区：
文献类型：
--
作者：
Beauchemin, C;Bougie, V;Laliberté, JF
为了开发一种在植物中生产外源蛋白的生物技术工具，我们首先设计了一种感染性芜菁花叶病毒（TuMV）cDNA，其包含水母绿色荧光蛋白（GFP）基因或细菌β-葡萄糖醛酸酶（GUS）基因（uidA）。评估了两个插入位点，在PI和HCPro顺反子之间或Pol和CP顺反子之间。在每个构建体中，插入基因侧翼的连接编码PI和/或VPg-Pro切割识别位点序列，以产生游离GUS或GFP。用基因枪转染黄绿芥后，出现TuMV感染的特征性症状，Westem blot分析表明病毒多聚蛋白中的GFP和GUS已被切除。在两个插入位点之间没有观察到表达水平的显著差异。通过RT-PCR，发现gfp在转染后30天（dpt）内是稳定的，而uidA在15 dpt时逐渐丢失。我们还创建了两个构建体，在同一分子上的每个插入位点含有任一基因。粒子轰击B后，观察到全身症状减轻。perviridis和Western印迹分析表明，两种外源蛋白都产生了。此外，观察到与单基因构建体相同的稳定性/不稳定性。这些结果表明，在基于TuMV的载体中同时产生至少两种外源蛋白是可能的。(c)2005 Elsevier B. V.保留所有权利。
With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertion sites were assessed, either between PI and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for PI and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Westem blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector. (c) 2005 Elsevier B.V. All rights reserved.