在尼科蒂亚娜本 thamiana中瞬态表达IgG融合蛋白的生产
1Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute,Arizona State University, 2School of Life Sciences,Arizona State University, 3Molecular Biosciences/Biotechnology Undergraduate Program,Arizona State University, 4Department of Microbiology and Immunology,University of Michigan Medical School
Summary
我们在这里描述了一个简单的方法，表达，提取和纯化重组人类IgG融合到GGFP 在尼科蒂亚娜本thamiana。该协议可以扩展到使用柱色谱的众多蛋白质的纯化和可视化。此外，该协议还适用于本学院教学实验室的亲上和虚拟教学实验室，提供基于项目的探索。
Abstract
对抗体作为各种传染性、代谢性、自身免疫性、肿瘤性和其他疾病的治疗干预措施的高需求，对开发有效的重组抗体生产方法的需求日益增长。截至2019年，有70多个FDA批准的单克隆抗体，并且有指数增长的潜力。尽管他们承诺，广泛使用的限制因素是制造成本和复杂性。工厂可能提供低成本、安全和易于扩展的蛋白质制造策略。像 Nicotiana benthamiana 这样的植物不仅能够正确折叠和组装复杂的哺乳动物蛋白质，还可以添加与哺乳动物细胞培养物类似的关键的翻译后修饰。在这项工作中，通过使用原生GP和绿色荧光蛋白（GFP）与人单克隆抗体融合的酸稳定变种，我们能够可视化来自 N.benthamiana 植物的整个瞬态抗体表达和纯化过程。根据实验的目的，本机 GFP 融合可以确保在植物的表达阶段更容易可视化，而酸稳定的 GFP 融合允许在下游处理期间实现可视化。这种可扩展和直接的程序可以由单个研究人员执行，只需几天就使用少数小植物就产生毫克量的高纯抗体或抗体融合蛋白。这种技术可以扩展到任何类型的抗体纯化过程和潜在的许多其他蛋白质的可视化，无论是植物和其他表达系统。此外，这些技术可以有利于虚拟指令，并在教学实验室由拥有分子生物学技术经验最少的本科生在教学实验室中执行，为基于项目的探索和实际应用奠定了基础。
Introduction
行业报告显示，在美国20种最毛额的药物中，有13种是生物制剂（蛋白质为基础的药物），其中9种是抗体。截至2019年，在1、2、3等不同临床开发阶段，有570多种抗体（Ab）治疗。目前全球Ab销售额超过1000亿美元，到2025年1月4日，单克隆Ab（mAb）治疗市场有望创造高达3000亿美元。由于需求如此之高，预计收入将增加，研究人员一直在努力开发各种方法，以更高的质量和更低的成本，在更大范围下生产Ab疗法。植物表达系统比传统的哺乳动物细胞系具有若干优势，可以负担得起和大规模制造Ab治疗剂5，6。在植物中生产蛋白质疗法（”分子法基”）不需要像传统的哺乳动物细胞培养技术7、8那样需要昂贵的生物反应器或细胞培养设施。植物不能收缩人类病原体，最大限度地减少潜在的污染。瞬态和转基因植物蛋白表达都可以作为哺乳动物或细菌生产系统的低成本替代品。虽然转基因植物是作物生产的首选，但使用这种方法重组蛋白质的生产可能需要数周到数月的时间。使用病毒载体通过注射器或真空农业渗透的瞬态表达方面的进步，允许在第11天、第12天、第13天、第14天分别对所需的蛋白质进行小规模和大规模生产。在N.Benthamiana植物15、16、17、18、19中，利用瞬态表达，生产了埃博拉、登革热和寨卡和许多其他重组蛋白的mAbs。这些情况使得瞬态植物表达成为开发多种Ab疗法的有吸引力的选择，并且本协议20中演示的方法。
第一代mAbs是喃自语衍生的，在人体试验21中使用时导致非特异性免疫原性。随着时间的推移，嵌合，人性化，并最终，完全人类Abs产生，以减轻免疫原性诱导Ab治疗。不幸的是，其中一些Abs仍然导致宿主免疫原性，由于糖基化21的差异。植物工程的发展允许对Ab甘油进行改造，这一点至关重要，因为Ab的稳定性和功能可能受到其糖化状态22的显著影响。进步使得植物系统中能够生产出含有人类甘油的高水平表达，并因此产生了大量生产的人类药物19、21所需的生物特性。
近几十年来，除了重组Abs之外，Ab聚变分子（Ab融合）还被探索用于各种目的。Ab融合通常由融合到分子或蛋白质的Ab或Ab片段组成，旨在引起免疫效应细胞23的反应。这些分子已被创建为潜在的治疗干预措施，以治疗各种病理，如癌症和自身免疫性疾病24，25，26，27。重组免疫复合物（RICs）是另一类Ab融合，已被采用作为疫苗候选28。RIC利用免疫系统识别Ab融合的Fc区域的能力，并被发现与其他疫苗平台29，30，31相结合，以提高免疫原性。
绿色荧光蛋白（GFP）是一种生物发光蛋白，源自维多利亚水母，当被紫外线照射时发出绿光32，33。多年来，GFP作为基因表达的视觉标记的使用已经从大肠杆菌的表达扩展到许多蛋白质表达系统，包括N.Benthamiana植物34，35，36，37，38。可见标记，如GP，对科学概念的教学和学习有着丰富的意义。许多入门级学生描述在所教导的想法肉眼看不见时，如分子生物学的概念和相关领域39时，掌握科学概念的困难。因此，视觉标记（如GP）有助于处理与科学过程有关的信息，并有助于减轻学生报告学习许多科学概念时遇到的困难。
虽然GP经常被用作体内指示基因和表达的标记物，但如果使用酸性条件，很难在下游过程中显示它。这种情况的主要原因是GP不保持其结构和由此产生的荧光在低pH40。在各种纯化过程中，通常需要临时酸性环境，如蛋白质G、蛋白质 A和蛋白质L色谱，通常用于Ab纯化41、42、43、44。GFP突变体已用于在酸性条件下保持荧光45，46。
在这里，我们描述了一个简单的方法，用于表达、提取和纯化重组IgG融合 蛋白在N.Benthamiana植物 。我们生产传统的GP融合到人性化IgG重链的N术语，创造了一个GP-IgG融合。同时，我们开发了一种植物共融优化序列的融合，用于酸性稳定 GFP （asGFP） 与人性化 IgG 重链的 N 术语，从而创建 asGFP-IgG 融合。生产GFP-IgG的优点包括能够在表达过程中可视化目标蛋白的存在，而SigFP-IgG则允许在表达和提取步骤中，而且在蛋白质的纯化步骤中看到重组蛋白的存在。该协议可用于生产、纯化和可视化一系列在 N. benthamiana 中生产的 GFP 融合蛋白， 并使用需要低 pH 值的色谱技术进行纯化。该工艺还可以针对各种数量的叶料进行定制。虽然用GFP或 asGFP 标记的 Abs 和融合蛋白并不可用于治疗，但这些方法在实验期间可用作控制，还可以作为分子和细胞生物学和生物技术的教学工具，无论是亲自还是虚拟。
Protocol
1. 培育 N. 本特塔米亚纳植物 将土壤泥炭颗粒放在托盘上，将以前煮沸、仍然热（+40-45 °C）的泥浆倒在泥炭颗粒上，进行完全膨胀。颗粒完全膨胀后，使用钳子将 2-3 N. 本特纳米纳种子放在每个泥炭颗粒上。 倒入约0.5入水以盖住托盘底部。将托盘与播种日期标记。继续每天用适当数量的肥料给幼苗浇水。肥料（水溶性多用途植物食品）浓度一?…
Representative Results
这项研究演示了一种简单而快速的方法，可以产生重组蛋白，并在整个下游过程中可视化它们。使用N. benthamiana并遵循所提供的协议，可在不到一周内实现此处描述的重组蛋白生产。植物表达、提取和纯化的总体工作流程如图1所示。图1 A（1-3）显示了2周大幼苗、4周岁幼苗和6周岁幼苗的生长阶段，而图1B则描绘了因</s…
Discussion
该协议可用于视觉验证在N.Benthamiana植物生产的任何重组Ab或重组蛋白，包括那些需要暂时暴露在酸性环境中用于柱纯化目的42，43，44。此外，ASGFP与不同表达系统中的其他蛋白质的融合是实验可视化和教育的有用工具。此处的协议可以进一步缩放到更大和更小的叶材料，以产生所需的重组蛋白量。所述方法利用了先前的?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢玛丽亚皮娅迪帕尔玛编辑的视频。我们还感谢亚利桑那州立大学教育外联和学生服务办公室慷慨的出版费援助。该议定书的研究得到了亚利桑那州立大学生命科学学院的支持。
Materials
| 5 mL syringe | any | N/A | |
| 50 mL syringe | any | N/A | |
| Agar | SIGMA-ALDRICH | A5306 | |
| Blender with cups | any | N/A | |
| Bromophenol blue | Bio-Rad | 1610404 | |
| DTT (DL-Dithiothreitol) | MP BIOMEDICALS | 219482101 | |
| EDTA (Ethylenedinitrilo)tetraacetic acid | SIGMA-ALDRICH | E-6760 | |
| Ethanol | any | N/A | |
| Glycerol | G-Biosciences | BTNM-0037 | |
| Glycine | SIGMA-ALDRICH | G7126-500G | |
| HCl (Hydrochloric acid) | EMD MILLIPORE CORPORATION | HX0603-4 | |
| Heating block | any reputable supplier | N/A | |
| Jiffy-7 727 w/hole peat pellets | Hummert International | 14237000 | |
| Kanamycin | Gold Biotechnology Inc | K-120-100 | |
| KCl (Potassium Chloride) | SIGMA-ALDRICH | P9541-500G | |
| KH2PO4 (Potassium Phosphate) | J.t.baker | 3248-05 | |
| KOH (Potassium Hydroxide) | VWR | BDH0262 | |
| Magnesium sulfate heptahydrate | SIGMA-ALDRICH | M2773 | |
| MES (2-(N-Morpholino)ethanesulfonic acid) | SIGMA-ALDRICH | M8250 | |
| Miracloth | Millipore | 4 75855-1R | |
| Moisture control potting mix | Miracle-Gro | 755783 | |
| Na2HPO4 (Sodium Phosphate) | J.t.baker | 3827-01 | |
| NaCl (Sodium Chloride) | Santa Cruz Biotechnology | sc-203274C | |
| Nicotiana benthamiana seeds | any reputable supplier | N/A | |
| PMSF (Phenylmethylsulfonyl Fluoride) | G-Biosciences | 786-787 | |
| Polypropylene Column | any | N/A | |
| Precision Plus Protein Dual Color Standards | Bio-Rad | 1610394 | |
| Protein G resin | Thermo Fisher Scientific | 20399 | |
| Rifampicin | Gold Biotechnology Inc | R-120-25 | |
| SDS (Sodium Dodecyl Sulfate) | G-Biosciences | DG093 | |
| Sodium Ascorbate | SIGMA-ALDRICH | A7631-500G | |
| Spectrophotometer | any reputable supplier | N/A | |
| Titan3 0.75 µm glass fiber filter | ThermoScientific | 40725-GM | |
| Tray for peat pellets with dome | any | N/A | |
| TRIS Base | J.t.baker | 4109-02 | |
| Tris-HCl | Amresco | M108-1KG | |
| Tryptone | SIGMA-ALDRICH | 17221 | |
| UV lamp | any | N/A | |
| Water Soluble All Purpose Plant Food | Miracle-Gro | 2000992 | |
| Yeast extract | SIGMA-ALDRICH | 9182 |
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