Name: transient expression in red beet of a biopharmaceutical candidate vaccine for type-1 diabetes
Uploaded: 2019-03-19T15:00:00
Description: Watch this Scientific Journal Video about Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes at JoVE.com

这项工作得到了维罗纳大学在2014年呼吁框架内资助的 "利用植物生产自身免疫性糖尿病食用疫苗" (项目 ID:891854) 联合项目的支持。

1. 红甜菜和菠菜栽培
<ol><li>生长红甜菜(b. vulgaris cv红磨坊) 和菠菜 (s.深恶痛绝 cv industria) 植物在生长室中, 分别使用150μe 的光强, 65% 相对湿度, 在231°c 下12小时光/暗周期。</li>
	<li>种子萌发后, 每周用市售肥料的 1 gl 溶液 (材料表) 给植物施肥两次。对于农业渗透使用5周龄菠菜和6周龄的红甜菜植物。</li>
</ol>2. 解构植物病毒技术的瞬态表达
<ol><li>植物?...

<ol>
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D., Patel, A., Boddu, S. H. S., Mitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+enhancing+oral+bioavailability+of+peptides+and+proteins.">Approaches for enhancing oral bioavailability of peptides and proteins.</a> International Journal of Pharmaceutics. 447, 75-93 (2013).</li><li>. Encapsulation importance in pharmaceutical area, how it is done and issues about herbal extraction Available from: <a target="_blank" href="https://www.researchgate.net/publication/271702091_Encapsulation_importance_in_pharmaceutical_area_how_it_is_done_and_issues_about_herbal_extraction">https://www.researchgate.net/publication/271702091_Encapsulation_importance_in_pharmaceutical_area_how_it_is_done_and_issues_about_herbal_extraction</a> (2015)</li><li>Kamei, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexation+hydrogels+for+intestinal+delivery+of+interferon+beta+and+calcitonin.">Complexation hydrogels for intestinal delivery of interferon beta and calcitonin.</a> Journal of Controlled Release. 134, 98-102 (2009).</li><li>Tuesca, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexation+hydrogels+for+oral+insulin+delivery:+effects+of+polymer+dosing+on+in+vivo+efficacy.">Complexation hydrogels for oral insulin delivery: effects of polymer dosing on in vivo efficacy.</a> Journal of Pharmaceutical Sciences. 97, 2607-2618 (2008).</li><li>Twyman, R. M., Schillberg, S., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+yield+of+recombinant+pharmaceutical+proteins+in+plants.">Optimizing the yield of recombinant pharmaceutical proteins in plants.</a> Current Pharmaceutical Design. 19, 5486-5494 (2013).</li><li>Dhama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-based+oral+vaccines+for+human+and+animal+pathogens+&#8211;+a+new+era+of+prophylaxis:+current+and+future+perspectives.">Plant-based oral vaccines for human and animal pathogens &#8211; a new era of prophylaxis: current and future perspectives.</a> Journal of Experimental Biology and Agricultural Sciences. 447, 75-93 (2013).</li><li>Hefferon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconceptualizing+cancer+immunotherapy+based+on+plant+production+systems.">Reconceptualizing cancer immunotherapy based on plant production systems.</a> Future science. O3, (2017).</li></ol>

在这项研究中, 我们对自身免疫性糖尿病候选口服疫苗的设计进行了初步分析。本实验的目标蛋白是人类 65 kDa 谷氨酸脱羧酶的突变形式, 其生产和功能易于检测和测量 12.它在不同食用植物组织中的表达是由载体5介导的, 在很短的时间内介导高水平的重组蛋白产生。根据红甜菜和菠菜叶片中 eGFP 表达的方法, 通过人工农业浸润, 选择了最佳候选植物食用寄主。由?...

自20世纪80年代植物分子生物学革命以来, 以植物为基础的生物制药生产系统可以被认为是基于微生物和哺乳动物细胞1 的传统系统的替代品。与传统平台相比, 工厂具有多种优势, 可扩展性、成本效益和安全性是最相关的 2。重组产物可以从转化后的植物组织中纯化, 然后在肠外或口服, 此外, 转化后的食用植物可以直接用于口服。口腔途径同时促进黏膜和全身免疫, 消除了对针头和专业医务人员的需求。此外, 口服可消除复杂的下游加工, 而下游加工通常占重组蛋白3制造总成本的80%。所有这些优势都可以转化为在生产、供应和劳动力方面的节约, 降低每剂的成本, 使全球大多数人口都能负担得起这种药物。
为植物重组蛋白的生产, 提出了几种稳定转化和瞬态表达的策略。其中, 一种高产量解构的植物病毒表达系统 (如 Mamonicon) 提供了卓越的性能, 在相对较短的时间范围内提高了重组蛋白的高产量4。报道了在作为金标准生产宿主的烟碱植物中使用植物病毒表达系统的许多瞬态表达的例子。然而, 这种示范植物并不被认为是一种可食用的物种, 因为它的叶子中积累了生物碱和其他有毒代谢物。
在这项工作中, 我们描述了两个可食用的植物系统的比较, 红色甜菜 (贝塔寻常的 Cv红磨坊) 和菠菜 (菠菜就是cv 工业), 表达了两种候选形式的 65 kda 谷氨酸异形脱羧酶 (GAD65), 由基于植物病毒的载体5进行。GAD65 是一种与1型糖尿病 (T1D) 相关的主要自身抗原, 目前正在人体临床试验中进行研究, 以通过诱导耐受性6来预防或延缓 T1D。在植物中生产 gad65 的过程中, 以烟叶和5%、6、7为典型植物种类进行了广泛的研究。在这里, 我们描述了使用食用植物物种生产的分子组织, 可用于直接口服。从技术角度出发, 通过对重组蛋白表达水平、植物残留微生物电荷等不同参数的评价, 研究并选择了 GAD65 生产的植物农业渗透系统和食用植物平台。用于口服的组织, GAD65 对胃消化的抵抗力, 以及转化后的植物与野生类型的生物等效性。

<table>
	<tbody>
		<tr>
			<td>0.2-&#956;m Minisart RC4 membrane filters</td>
			<td>Sartorius-Stedim</td>
			<td>17764</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#8211;mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td>Toxic; 4 % to make loading buffer with glycerol, SDS and Tris-HCl</td>
		</tr>
		<tr>
			<td>4-Morpholineethanesulfonic acid (MES)</td>
			<td>Sigma</td>
			<td>M8250</td>
			<td>pH 5.5</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sarstedt</td>
			<td>833924</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>27221</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Acetonitrile LC-MS grade</td>
			<td>Sigma</td>
			<td>34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
			<td>Toxic &#8211; 0.1 M stock in DMSO</td>
		</tr>
		<tr>
			<td>Agar Bacteriological Grade</td>
			<td>Applichem</td>
			<td>A0949</td>
			<td>15 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Tryptone</td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Sigma</td>
			<td>70221</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-eGFP antibody</td>
			<td>ABCam</td>
			<td>ab290</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAD 65/67 antibody</td>
			<td>Sigma</td>
			<td>G5163</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-LHCB2 antibody</td>
			<td>Agrisera</td>
			<td>AS01 003</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Blue R-250</td>
			<td>Sigma</td>
			<td>B7920</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Alltima HP C18 (150 mm x 2.1 mm; 3 &#956;m) Column</td>
		</tr>
		<tr>
			<td>C18 Guard Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Alltima HP C18 (7.5 mm x 2.1 mm; 5 &#956;m) Guard&#160; Column</td>
		</tr>
		<tr>
			<td>CalMag Grower</td>
			<td>Peter Excel</td>
			<td>15-5-15</td>
			<td>Fertilizer</td>
		</tr>
		<tr>
			<td>Carbenicillin disodium</td>
			<td>Duchefa Biochemie</td>
			<td>C0109</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Chemiluminescence imaging system</td>
			<td>BioRad</td>
			<td>1708370</td>
			<td>ChemiDoc Touch Imaging System</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent</td>
			<td>Sigma</td>
			<td>P5927</td>
			<td>Polysorbate 20</td>
		</tr>
		<tr>
			<td>Fluorescence reader</td>
			<td>Perkin-Elmer</td>
			<td>&#160;1420-011</td>
			<td>VICTOR Multilabel Counter</td>
		</tr>
		<tr>
			<td>Formic acid LC-MS grade</td>
			<td>Sigma</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>15 % to make loading buffer with Tris-HCl, SDS and 2&#8211;mercaptoethanol</td>
		</tr>
		<tr>
			<td>GoTaq G2 polymerase</td>
			<td>Promega</td>
			<td>M7841</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>HILIC Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Ascentis Express HILIC (150 mm x 2.1 mm; particles size 2.7 &#956;m) Column</td>
		</tr>
		<tr>
			<td>HILIC Guard Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Vision HT HILIC (7.5 mm x 2.1 mm; 3 &#956;m) Guard&#160; Column</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td>
			<td>Sigma</td>
			<td>A6154</td>
			<td>Do not freeze/thaw too many times</td>
		</tr>
		<tr>
			<td>HPLC Autosampler</td>
			<td>Beckman Coulter</td>
			<td>&#160;&#160; -</td>
			<td>System Gold 508 Autosampler</td>
		</tr>
		<tr>
			<td>HPLC System</td>
			<td>Beckman Coulter</td>
			<td>&#160;&#160; -</td>
			<td>System Gold 128 Solvent Module HPLC</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>24137</td>
			<td>Flamable</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Sigma</td>
			<td>K4000</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>2 g/L with NaCl , Na2HPO4 and KH2PO4 to make PBS</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P9791</td>
			<td>2.4 g/L with NaCl , Na2HPO4 and KCl to make PBS</td>
		</tr>
		<tr>
			<td>Loading Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol solution</td>
			<td>Ge Healthcare</td>
			<td>RPN2232</td>
			<td>Prepare the solution using the ECL Prime Western Blotting System commercial kit</td>
		</tr>
		<tr>
			<td>Lyophilizator</td>
			<td>5Pascal</td>
			<td>LIO5P0000DGT</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectometer</td>
			<td>Bruker Daltonics</td>
			<td>&#160; -</td>
			<td>Bruker Esquire 6000; the mass spectrometer was equipped with an ESI source and the analyzer was an ion trap</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>32213</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk-blocking solution</td>
			<td>Ristora</td>
			<td>&#160;&#160; -</td>
			<td>3 % in PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td>Use with NaH2PO4 to make Sodium Phospate buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>80 g/L with KCl, Na2HPO4 and KH2PO4 to make PBS; 10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, Tryptone and Agar Bacteriological Grade</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>&#160;Use with Na2HPO4 to make Sodium Phospate buffer; 14.4 g/L to make PBS</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulase membrane</td>
			<td>Ge Healthcare</td>
			<td>10600002</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin from porcine gastric mucosa</td>
			<td>Sigma</td>
			<td>P7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase substrate ECL</td>
			<td>GE Healthcare</td>
			<td>RPN2235</td>
			<td>Light sensitive material</td>
		</tr>
		<tr>
			<td>Pump Vacuum Press</td>
			<td>VWR</td>
			<td>111400000098</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent A</td>
			<td>Sigma</td>
			<td>B9643</td>
			<td>Use 50 parts of this reagent with 1 part of reagent B to prepare BCA working solution</td>
		</tr>
		<tr>
			<td>Reagent B</td>
			<td>Sigma</td>
			<td>B9643</td>
			<td>Use 1 part of this reagent with 50 parts of reagent A to prepare BCA working solution</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Duchefa Biochemie</td>
			<td>R0146</td>
			<td>Toxic &#8211; 25 mg/mL stock in DMSO</td>
		</tr>
		<tr>
			<td>SDS (Sodium dodecyl sulphate)</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td>Flamable, toxic, corrosive-10 % stock; 3 % to make loading buffer with Tris-HCl, Glycerol and 2&#8211;mercaptoethanol</td>
		</tr>
		<tr>
			<td>Sodium metabisulphite</td>
			<td>Sigma</td>
			<td>7681-57-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator system</td>
			<td>Soltec</td>
			<td>090.003.0003</td>
			<td>Sonica&#174; 2200 MH; frequency 40 khz</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Terumo</td>
			<td>&#160;&#160; -</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent fixed 300-&#181;L insert glass tubes</td>
			<td>Thermo Scientific</td>
			<td>11573680</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Adjust pH with 1N HCl to make Tris-HCl buffer, use 1,5M Tris-HCl (pH 6.8) to make loading buffer with SDS, Glycerol and 2&#8211;mercaptoethanol</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Formedium</td>
			<td>TRP03</td>
			<td>10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Agar Bacteriological Grade</td>
		</tr>
		<tr>
			<td>Vacuum concentrator</td>
			<td>Heto</td>
			<td>3878 F1-3</td>
			<td>Speed-vac System</td>
		</tr>
		<tr>
			<td>Water LC-MS grade</td>
			<td>Sigma</td>
			<td>39253</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma</td>
			<td>Y1333</td>
			<td>5 g/L to make LB medium (pH 7.5 with NaOH) with Tryptone, NaCl and Agar Bacteriological Grade</td>
		</tr>
	</tbody>
</table>

transient expression in red beet of a biopharmaceutical candidate vaccine for type-1 diabetes

植物分子耕作是利用植物生产感兴趣的分子。从这个角度来看, 植物既可以作为生物反应器, 用于生产和随后纯化最终产品, 也可以用于使用可食用植物物种时直接口服异源蛋白质。在这项工作中, 我们提出了一个候选口服疫苗1型糖尿病 (T1D) 在食用植物系统中使用解构植物病毒重组 DNA 技术, 以真空渗透交付。我们的研究结果表明, 红甜菜是一个合适的宿主, 用于与 T1D 相关的人类衍生自身抗原的瞬态表达, 被认为是 T1D 疫苗的一个有希望的候选疫苗。对产生自体抗原的叶片进行了彻底的检测, 以了解其对胃消化的抵抗力、残留细菌电荷的存在以及对其的次生代谢状况, 并概述了该过程中的生产, 以便对其潜在的用途进行概述用于直接口服异源蛋白的植物。我们的分析显示, 在模拟胃消化后, 冻干候选口服疫苗几乎完全降解, 这表明在制造植物衍生的 GAD 疫苗时需要一种封装策略。

本文介绍了在食用植物组织中开发口服疫苗的工作流程。这项工作的重点是在可食用寄主植物物种中表达目标蛋白和对潜在口服疫苗的定性。
第一步是评估基于植物病毒的表达技术是否适合在食用植物系统中产生重组蛋白。为此, 我们首先使用 eGFP 作为模型蛋白, 并将其表达为两个可食用的叶植物系统: 红甜菜和菠菜。植物通?...

Watch this Scientific Journal Video about Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes at JoVE.com

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

1型糖尿病生物制药候选疫苗在红甜菜中的瞬态表达

在这里, 我们提出了一个协议, 以产生一个口服疫苗候选1型糖尿病在食用植物。

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

在植物中生产重组蛋白的不同策略中，解构的基于植物的VERSS表达提供卓越的性能，在相对较短的时间范围内产生高产量。复制这一技术以生产口服疫苗具有在不久的将来成为传统疫苗替代品的巨大潜力。2月冻红甜菜叶瞬息万变积累大量适合口服耐受诱导的光抗原，预防1型糖尿病。在对重组蛋白积累进行个案评估后，该实验方案可以扩展到许多不同的食用物种。演示手术的将是爱德华多·贝尔蒂尼、马蒂娅·桑托尼、安娜·库库鲁洛和罗伯塔·赞皮耶里。要开始此过程，请像文本协议中所述，在生长室中种植红甜菜和菠菜植物。种子发芽后，每周用市售肥料溶液施肥两次，浓度为每升一克。对于农业过滤，使用五周大的菠菜和六周大的红甜菜植物。在构建植物表达载体后，在含有利福平的50毫升LB介质中接种三种 A.tumefaciens 转化剂，浓度为每毫升50微克，以及文本协议中概述的病媒特异性抗生素。在28摄氏度下一夜之间摇晃生长。第二天，在4500次G下离心20分钟，对细菌培养物进行颗粒化。将颗粒重新注入含有 10 毫摩尔 MES 和 10 毫摩尔硫酸镁的 100 毫升渗透缓冲液中。在室温下孵育悬浮液三小时。将包含此处所示的模块之一的细菌悬浮液的等量与五个质模块和整数模块混合。使用无针头的注射器，拿出五毫升的悬浮液混合物。将注射器的尖端压在植物叶的底面上，同时轻轻对叶的另一侧施加反压。渗透到前三个完全膨胀的叶子，从顶点开始，每个植物。在叶茎上贴上纸标签，标记农业渗透的叶子。然后在标准条件下将植物返回生长室。感染后的第4至14天，收集农业渗透的叶子，并在液氮中冷冻。将这种植物组织储存在零下80摄氏度。首先，在50毫升的LB介质中分别种植三种 A.tumefacien 转化剂，其浓度为每毫升50微升，并在28摄氏度下过夜摇动，并提供适当的病媒特异性抗生素。第二天，在4500次G下离心20分钟，对细菌培养物进行颗粒化。将颗粒在 1 升渗透缓冲液中重新悬浮到 0.35 的 OD600 中，并在室温下孵育悬浮液三小时。然后在每个悬浮液中加入0.01%的洗涤剂。将包含此处所示的模块之一的细菌悬浮液的等量与五个质模块和整数模块混合。将一个六周大的红色甜菜植物插入支架。倒置支架，并放在装有两升渗透浴的烧杯上，将叶子淹没在渗透悬浮液中。之后，将浸入式植物的渗透浴转移到渗透室并关闭。打开真空泵并打开渗透室上的真空进气阀。一旦腔室中的压力下降到 90 毫巴，保持真空三分钟。然后释放真空 45 秒。当腔室回到大气压力时，打开腔室，将渗入的植物从细菌浴中清除。将工厂返回到生长室。在表达最大一天，收获渗透的叶子，并冻结在液氮，如前面所述。首先，在表达高峰期收获真空农业渗透三角洲87GAD65mut表示红甜菜叶，并在液氮中冷冻。将冷冻叶在零下50摄氏度和0.04毫巴下冻干72小时。然后将它们储存在零下80摄氏度。准备好继续操作时，将叶子研磨成细粉末，并在室温下存放在密封的容器中，并含有硅胶，以排除水分。对于胃消化模拟，重达100毫克的细磨冷冻干燥的红甜菜叶，并重新暂停在6毫升PBS。使用六摩尔盐酸将样品pH值调整为2。从猪胃粘膜中加入四毫克每毫升辣椒，加入 10 毫摩尔盐酸，以获得每毫升一毫克的最终百事可乐浓度。在 37 摄氏度下摇动样品 120 分钟。接下来，使用氢氧化钠将样品pH值调整为8，并灭活辣椒。将每个样品的750微升等分转移到微离心管中，并在20，000倍G和4摄氏度下将等分离离心20分钟。分别收集每个上一时分，并在一个上流负载缓冲器体积中重新暂停每个颗粒。然后通过西方印迹分析分析上重物和再增颗粒。对于细胞完整性分析，准备两个100毫克的细磨冻干红甜菜叶的样品，并在PBS的6毫升中重新暂停。使用六摩尔盐酸将一个样品的 pH 值调整为两个。在37摄氏度下摇动两个样品120分钟。然后将每个样品的750微升分到微离心管。在20，000倍G和4摄氏度的离心机20分钟。分别收集每个上一时分，并在一个上流负载缓冲器体积中重新暂停每个颗粒。然后通过西方印迹分析分析上重物和再增颗粒。首先，重达100毫克的细磨冻干红甜菜叶，并重新暂停在8毫升无菌PBS。漩涡一分钟。在五种准备好的选择性 LB 介质中，每片冻干叶均质的一毫升板。在28摄氏度下孵育板三天。在此之后，计算每个板上生长的农业菌菌群，并计算残留细菌电荷为冻干叶同质化每毫升形成单位的数量。在这项工作中，在可食用植物组织中研制出口服疫苗。红甜菜和菠菜植物被手工农业渗透，悬挂的A.tumefacien携带EGFP重组表达载体，荧光蛋白表达通过西方印迹分析可视化，并在紫外线下进行量化。红甜菜系统的特点是EGFP表达较高，感染后9天达到每克新鲜叶体重约544微克，而菠菜在感染后11天仅达到每克新鲜叶体重约113.4微克的最大值。因此，红色甜菜被选为所有后续实验的表达式宿主。然后，植物被真空渗透与 A.tumefaciens 悬浮液。从农业渗透叶中提取TSP后，用西方印迹对样品进行分析，通过密度测定分析对重组蛋白进行相对量化。最后，对潜在口服疫苗的研制参数进行了评价。如这里看到，目标蛋白在冻干过程后保持稳定。通过将猪胃酶肽加入冷冻干燥物质，或将最终浓度为每毫升1毫克，或与TSP的1至20比比，模拟胃消化。两种消化治疗条件都会导致重组蛋白降解。在肽消化后颗粒样品中缺乏特定信号表明，冷冻干燥后，植物细胞失去了完整性，这导致了目标蛋白的降解。在真空渗透方面，应仔细适应植物物种和目标重组蛋白的时间过程分析。示范程序也可用于生产用于口服分娩的生物制药，如疫苗和抗体。

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

创建瞬态细胞膜毛孔，使用标准的喷墨打印机

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

微流控控制环境的红细胞聚集动态调查

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...