High-level expression and enrichment of norovirus virus-like particles in plants using modified geminiviral vectors
High-level expression and enrichment of norovirus virus-like particles in plants using modified geminiviral vectors
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DOI：
10.1016/j.pep.2018.06.011
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发表时间：
2018-11-01
影响因子：
1.6
通讯作者：
Mason, Hugh S.
中科院分区：
文献类型：
--
作者：
Diamos, Andrew G.;Mason, Hugh S.
重组病毒样颗粒（VLP）已被证明是安全有效的候选疫苗。我们先前已经描述了一种基于植物的重组蛋白表达系统，该系统基于来自双生病毒属菜豆黄矮病毒（BeYDV）的复制载体的农杆菌渗入。该系统已被系统优化，以提高表达和减少本氏烟草叶中的细胞死亡。使用这些修饰，我们表明，来自基因型GII.4诺如病毒（全球急性胃肠炎的主要原因）的VLP可以以> 1 mg/g叶鲜重（LFW）产生，是基于植物的系统中报道的最高水平的三倍以上。我们还生产了2.3mg/g LFW的诺如病毒GI VLP。用酸、洗涤剂或热处理含VLP的粗叶提取物提高了回收率，并允许选择性富集诺如病毒VLP。最佳处理条件允许去除> 90%的内源植物蛋白而没有任何诺如病毒VLP的损失。还证明了通过酸处理选择性富集乙型肝炎B核心抗原（HBcAg）VLP，在去污剂存在下部分减轻了产量的一些损失。沉降分析证实，酸和去污剂不抑制诺如病毒VLP的正确组装，尽管热处理具有小的负面影响。这些结果表明，使用BeYDV植物表达系统，可以在几天内从单个植物叶获得毫克量的诺瓦克病毒VLP并高度富集。
Recombinant virus-like particles (VLPs) are proven to be safe and effective vaccine candidates. We have previously described a plant-based recombinant protein expression system based on agroinfiltration of a replicating vector derived from the geminivirus bean yellow dwarf virus (BeYDV). The system has been systematically optimized to improve expression and reduce cell death in Nicotiana benthamiana leaves. Using these modifications, we show that VLPs derived from genotype GII.4 norovirus, the leading cause of acute gastroenteritis worldwide, can be produced at > 1 mg/g leaf fresh weight (LFW), over three times the highest level ever reported in plant-based systems. We also produced norovirus GI VLPs at 2.3 mg/g LFW. Treatment of VLP-containing crude leaf extracts with acid, detergent, or heat enhanced recovery and allowed selective enrichment of norovirus VLPs. Optimal treatment conditions allowed removal of > 90% of endogenous plant proteins without any loss of norovirus VLPs. Selective enrichment of hepatitis B core antigen (HBcAg) VLPs by acid treatment was also demonstrated, with some losses in yield that were partially mitigated in the presence of detergent. Sedimentation analysis confirmed that acid and detergent did not inhibit proper assembly of norovirus VLPs, although heat treatment had a small negative effect. These results demonstrate that milligram quantities of norovirus VLPs can be obtained and highly enriched in a matter of days from a single plant leaf using the BeYDV plant expression system.