Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells
Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells
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DOI：
10.1007/s00705-005-0705-8
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发表时间：
2006-06-01
影响因子：
2.7
通讯作者：
Mori, M.
中科院分区：
文献类型：
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作者：
Dohi, K.;Nishikiori, M.;Mori, M.
虽然悬浮培养的植物细胞作为有用蛋白质的来源具有许多潜在的优点，但缺乏有效的表达系统阻碍了这种方法的使用。在本研究中，我们建立了一个诱导型番茄花叶病毒（ToMV）感染烟草BY-2悬浮培养细胞的系统，以诱导和有效地产生外源蛋白。在该系统中，在转基因BY-2细胞中，在雌激素诱导型启动子的控制下，从稳定转化的cDNA表达编码外源蛋白作为外壳蛋白替代物的修饰ToMV。添加到培养物中的雌激素激活从转基因组成型表达的雌激素诱导型反式激活因子，并诱导病毒RNA的转录和复制。在我们的实验中，病毒RNA的积累和绿色荧光蛋白（GFP）编码的病毒在诱导后的24小时内观察。诱导后4d，GFP表达量达到可溶性总蛋白的10%左右。相反，在未诱导的细胞中既没有检测到病毒RNA也没有检测到GFP。建立的诱导型病毒感染系统不仅可用于外源蛋白的表达，而且可用于研究病毒在培养植物细胞中的复制过程。
Although suspension-cultured plant cells have many potential merits as sources of useful proteins, the lack of an efficient expression system has prevented using this approach. In this study, we established an inducible tomato mosaic virus (ToMV) infection system in tobacco BY-2 suspension-cultured cells to inducibly and efficiently produce a foreign protein. In this system, a modified ToMV encoding a foreign protein as replacement of the coat protein is expressed from stably transformed cDNA under the control of an estrogen-inducible promoter in transgenic BY-2 cells. Estrogen added to the culture activates an estrogen-inducible transactivator expressed constitutively from the transgene and induces transcription and replication of viral RNA. In our experiments, accumulation of viral RNA and expression of green fluorescent protein (GFP) encoded in the virus were observed within 24h after induction. The amount of GFP reached approximately 10% of total soluble protein 4d after induction. In contrast, neither viral RNA nor GFP were detected in uninduced cells. The inducible virus infection system established here should be utilized not only for the expression of foreign proteins, but also for investigations into the viral replication process in cultured plant cells.