School of Environmental Sciences, University of Guelph, Guelph, ON, Canada.
Plant Biotechnol J. 2012 Dec;10(9):1118-28. doi: 10.1111/j.1467-7652.2012.00742.x. Epub 2012 Sep 17.
To study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAi-based glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5' and 3' UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5'UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.
为了研究 P19 基因沉默抑制蛋白如何有效地用于治疗性糖蛋白的生产，我们考察了以下因素：表达重组蛋白的遗传元件；不同 P19 浓度的影响；P19 与各种烟草品种用于转基因表达的兼容性；与 P19 共表达的重组治疗性糖蛋白的聚糖谱在基于 RNAi 的糖基化修饰的烟草表达宿主中。曲妥珠单抗的重链和轻链编码序列被克隆到含有不同 5'和 3'UTR 的五个植物表达载体（102-106）中，命名为载体集 102-106 mAb。番茄丛矮病毒（TBSV）的 P19 蛋白也被克隆到载体 103 中，该载体含有花椰菜花叶病毒（CaMV）35S 启动子和 5'UTR 以及农杆菌的胭脂碱合成酶基因的终止子区域。抗体载体的瞬时表达导致曲妥珠单抗的积累水平不同，最高的是载体 105 和 106 mAb，约为 TSP 的 1%。当与 103 mAb 共表达时，P19 将曲妥珠单抗的浓度提高了约 15 倍（约为 TSP 的 2.3%），但对载体 102 和 106 mAb 的抗体水平没有影响。当 103 mAb 与 P19 在不同的烟草品种中共同表达时，除了小克里坦登（Little Crittenden）之外，所有品种的叶片浸润区域都明显变色，抗体表达减少。P19 的共表达也使烟草品种 I-64 和小克里坦登之间的杂交中抗体的积累消失，表明观察到的 P19 诱导的反应具有显性遗传模式。
