Collens Jason I, Mason Hugh S, Curtis Wayne R
Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biotechnol Prog. 2007 May-Jun;23(3):570-6. doi: 10.1021/bp060342u. Epub 2007 Apr 11.
A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector. All vectors were delivered to Nicotiana glutinosa plant cell suspension or hairy root cultures via auxotrophic Agrobacterium tumefaciens. Cell culture generated greater yield of reporter gene expression than did root culture, as a result of the limitation imposed on roots to express the protein only in surface tissue containing actively dividing cells. Reporter gene expression increased for cell culture when the reporter gene construct was co-delivered with the construct supplying both viral replication associated proteins (REP and REPA); gene expression decreased when the construct supplying only the viral REP protein was co-delivered. Reporter protein expression increased from 0.091% for the reporter construct alone to 0.22% total soluble protein (% TSP) when the viral Rep-supplying vector was co-delivered with the reporter gene construct. Reporter protein was generated 3 days after the initiation of bacterial co-culture, providing for rapid generation of heterologous protein in cell culture.
研究了一种基于菜豆黄矮病毒的病毒载体提高瞬时基因表达的潜力。将含内含子的GUS报告基因和顺式作用病毒调控元件整合到病毒载体中，并可由辅助载体上提供的病毒复制相关蛋白进行互补。所有载体均通过营养缺陷型根癌农杆菌导入烟草细胞悬浮液或毛状根培养物中。由于根只能在含有活跃分裂细胞的表面组织中表达蛋白质，细胞培养产生的报告基因表达量高于根培养。当报告基因构建体与提供病毒复制相关蛋白（REP和REPA）的构建体共同导入时，细胞培养中的报告基因表达增加；当仅提供病毒REP蛋白的构建体共同导入时，基因表达降低。当提供病毒Rep的载体与报告基因构建体共同导入时，报告蛋白表达从单独报告构建体的0.091%增加到总可溶性蛋白的0.22%（%TSP）。细菌共培养开始3天后产生报告蛋白，从而在细胞培养中快速产生异源蛋白。
