通过Bioorthogonal点击化学化学选择性病毒表面改性
Summary
腺病毒颗粒设计，含有非天然氨基酸类似物azidohomoalanine或叠氮糖<em>Ø</em> GlcNAz。每叠组chemoselectively结扎通过“点击”化学反应病毒表面改性的一种手段。
Abstract
病毒颗粒的修改已收到的关注显着，其影响的基因治疗，溶瘤应用和疫苗开发的巨大潜力。1,2,3修改病毒的表面，其中大多是遗传学的基础，当前的方法往往受到衰减生产病毒，传染性和细胞转导。4,5使用化学选择性的点击化学，我们已经开发出一个简单的替代方法回避这些问题，同时保持高度灵活和方便。1,2
本议定书的目标是展示使用bioorthogonal点击化学修改的5型腺病毒颗粒表面的成效。可用于同时治疗1或分析，2,6这两个步骤的过程，作为靶向分子，染料或其他分子的化学选择性结扎允许到蛋白质预先标记叠标签。这种方法的三个主要优点是：（1）代谢标记表明几乎没有病毒健身的影响，1,7（2）广泛的效应配体可以利用，（3）它是非常快速，可靠和易于访问。1,2,7
在此过程的第一步，腺病毒颗粒生产轴承要么azidohomoalanine（AHA，蛋氨酸的替代）或不自然的糖O型联的 N-azidoacetylglucosamine（ 邻 GlcNAz），它们都含有叠氮化物（氮3）功能组。净化后的叠改性病毒颗粒，包含：TAMRA荧光基炔探头结扎预标记的蛋白质或糖蛋白在化学选择性的方式。最后，进行SDS-PAGE分析证明到病毒衣壳蛋白探头成功结扎。阿哈纳入标记所有病毒衣壳蛋白（Hexon蛋白，聚氯和光纤），而O型 GlcNAz只标签纤维掺入结果。
在这个不断发展的领域，已成功开发出多个叠 – 炔结扎方法，但是只有两个我们已经找到了，是最方便的证明本 – 促进应变叠氮化物 – 炔环加成（SPAAC）和铜催化的叠炔环加成（CuAAC）在无氧气氛下。
Protocol
请参阅表1，准备在这个协议中引用的所有媒体，缓冲区和解决方案。 1。生产的AHA-标记的腺病毒准备人类胚胎肾脏细胞11 100毫米组织培养皿（HEK 293）（ 见表1）在HEK 293细胞的生长介质保持在37°C，直到他们达到80 – 90％汇合。 （注：文化的汇合90％以上的感染，可能不会产生所需的病毒可能很难。） 松开的菜肴之一，首先消除的生长介…
Discussion
在化学选择性和bioorthogonal点击反应，包括叠氮化合物的的发展是一个发展迅速的研究领域，以及随后出现的这些反应越来越多的选择，从生物耦合应用。我们已经限制了该协议的范围包括在我们自己的实验室和所有试剂的商业可用性的方法，因为它们的用途选择只有两个。
在第一个这样的路线-应变促进叠-炔环加成（SPAAC） -炔载内cyclooctyne环（ 图2a）。这是最近…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢国家科学基金会拨款（CBET-0846259）。
Materials
| Name of the reagent | Company | Catalogue number |
| Adenovirus type 5 (Ad5) containing a GFP transgene | BCBC | 391 |
| Human Embryonic Kidney (HEK 293) cells | ATCC | CRL-1573 |
| Dulbecco’s Modified Eagle Medium (DMEM), High Glucose | Invitrogen | 11965-092 |
| Dulbecco’s Modified Eagle Medium, no Methionine, no Cysteine (DMEM -Met / -Cys), High Glucose | Invitrogen | 21013-024 |
| Bovine Calf Serum | Invitrogen | 16170-078 |
| Penicillin – Streptomycin 100× Solution (Pen Strep) | Invitrogen | 15140-122 |
| 0.5% Trypsin-EDTA (10×) | Invitrogen | 15400-054 |
| 100 mm Cell Culture Dish, tissue-culture treated polystyrene | BD Falcon | 353003 |
| Cell culture CO2 incubator | ||
| Hemocytometer | ||
| Biosafety cabinet | ||
| Sterile syringe filter (0.2 μm, cellulose acetate) | VWR | 28145-477 (NA), 514-0061 (Europe) |
| Avanti J-E centrifuge | Beckman Coulter | 369001 |
| Conical tubes (50 ml, sterile) | BD Falcon | 352098 |
| Tube, Thinwall, Ultra-Clear, 13.2 ml, 14 x 89 mm | Beckman | 344059 |
| Ultracentrifuge equipped with an SW 41 and SW 60 rotor | Beckman | |
| Eppendorf Biopur Safe-Lock Tubes, 1.5 ml | Eppendorf | 0030 121.589 |
| Centrifuge 5418 | Eppendorf | 5418 |
| Centri-Sep gel filtration spin columns | Princeton Separations | CS-901 |
| Sterile needle, 18 gauge | ||
| Nitrogen glove bag (if deoxygenated CuAAC is to be performed) | ||
| Dewar flask | ||
| Liquid nitrogen | ||
| Electrophoresis cell | ||
| Fluorescent gel scanner | ||
| Ready Gel Tris-HCl Gel | Bio-Rad | 161-1105 |
| L-Azidohomoalanine | AnaSpec | 63669 |
| Jena Biosciences | CLK-AA005 | |
| Peracetylated N-azidoacetylgalactosamine (Ac4GalNAz) | Invitrogen | C33365 |
| Thermo Scientific | 88905 | |
| Sigma-Aldrich | A7480 | |
| Bathophenanthroline disulfonic acid (BDA) disodium salt | MP Biomedicals | 0215011201 |
| Dimethyl sulfoxide (DMSO) | ||
| Methanol | ||
| Tris base (tris(hydroxymethyl)aminomethane) | ||
| Disodium Phosphate (Na2HPO4) | ||
| Phosphate buffered saline (PBS) | ||
| 1M HCl | ||
| Glycerol | ||
| Bovine Serum Albumin | ||
| L-cysteine | ||
| L-methionine | ||
| SDS (sodium dodecyl sulfate) | ||
| 2-mercaptoethanol | ||
| Glycine | ||
| Bromophenol blue | ||
| Cesium Chloride (CsCl) | ||
| Copper(I) Bromide (CuBr) | ||
| Calcium Chloride (CaCl2) | ||
| Potassium Chloride (KCl) | ||
| Magnesium Chloride (MgCl2) | ||
| Sodium Chloride (NaCl) | ||
| Alkyne probe for CuAAC* | ||
| TAMRA Alkyne | Invitrogen | T10183 |
| Strained alkyne probe for SPAAC* | ||
| TAMRA DIBO Alkyne | Invitrogen | C10410 |
* Notable vendors of click chemistry reagents and kits include Invitrogen, Jena Biosciences, Berry Associates, Sigma-Aldrich, Glen Research, Click Chemistry Tools, and Baseclick. A variety of alkyne dyes and targeting ligands can be found in these vendors’ catalogs.
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Tags
Chemoselective ModificationViral SurfacesBioorthogonal Click ChemistryGene TherapyOncolytic ApplicationsVaccine DevelopmentGenetics-based ApproachesChemoselective LigationTargeting MoleculesDyesAzide TagsMetabolic LabelingEffector LigandsAdenovirus ParticlesAzidohomoalanineO-linked N-azidoacetylglucosamine
Cite This Article
Rubino, F. A., Oum, Y. H., Rajaram, L., Chu, Y., Carrico, I. S. Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry. J. Vis. Exp. (66), e4246, doi:10.3791/4246 (2012).