Centro Ricerche Casaccia, Via Anguillarese 301, I-00123, Rome, Italy.
BMC Biotechnol. 2009 Nov 20;9:96. doi: 10.1186/1472-6750-9-96.
In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.
The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue.
We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.
近年来，通过稳定转化或瞬时表达系统，不同的 HIV 抗原已成功在植物中表达。在 HIV 蛋白中，Nef 被认为是一种有前途的多组分疫苗靶点，因为它涉及病毒感染的第一步。试图将 Nef 作为一种单一的蛋白质产物（不与稳定蛋白融合）在转基因植物中表达，导致产量令人失望地低（约占总可溶性蛋白的 0.5%）。在这项工作中，我们描述了一种基于瞬时表达的系统，该系统基于在 Nicotiana benthamiana 中共同农杆菌浸润植物病毒基因沉默抑制蛋白，然后通过两步亲和纯化方案从植物中纯化衍生的 Nef。
评估了三种基因沉默病毒抑制蛋白（马铃薯 X 病毒的 P25、蓟花叶斑驳卷曲病毒和番茄丛枝矮化病毒的 P19）对 Nef 瞬时表达产量的影响。蓟花叶斑驳卷曲病毒的 P19 蛋白（AMCV-P19）在真空共农杆菌浸润实验中给出了最高的表达产量，达到总可溶性蛋白的 1.3%，几乎是以前在稳定转基因植物中报道的三倍。在共农杆菌浸润植物中观察到的高产量与 Nef 特异性小干扰 RNA（siRNA）的显著减少相关，表明 AMCV-P19 有效地调节了 RNA 沉默机制。有趣的是，我们还发现与底部叶片相比，真空共农杆菌浸润植物的顶部叶片中的表达水平明显降低。此外，通过两步固定化金属离子亲和层析方案从 agroinfiltrated 组织中纯化 Nef，产量为 250ng/g 新鲜组织。
我们证明，使用 AMCV-P19 基因沉默抑制蛋白增强的瞬时表达系统可以提高 HIV-1 Nef 在植物中的表达水平。此外，利用两步纯化方案，提高了产量，纯化了植物来源的 Nef。这些结果代表了开发植物源性 HIV 疫苗的第一步。
