Name: chemoselective modification of viral surfaces via bioorthogonal click chemistry
Uploaded: 2012-08-19T14:00:00
Description: Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

我们要感谢国家科学基金会拨款（CBET-0846259）。

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<ol><li>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。） </li><li>松开的菜肴之一，首先消除的生长介...

<ol>
	<li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoselective+Attachment+of+Small+Molecule+Effector+Functionality+to+Human+Adenoviruses+Facilitates+Gene+Delivery+to+Cancer+Cells.">Chemoselective Attachment of Small Molecule Effector Functionality to Human Adenoviruses Facilitates Gene Delivery to Cancer Cells.</a> J. Am. Chem. Soc. 132, 13615-13617 (2010).</li><li>Banerjee, P. S., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoselective+Modification+of+Viral+Proteins+Bearing+Metabolically+Introduced+"Clickable"+Amino+Acids+and+Sugars.">Chemoselective Modification of Viral Proteins Bearing Metabolically Introduced "Clickable" Amino Acids and Sugars.</a> Methods Mol. Biol. 751, 55-66 (2011).</li><li>Waehler, R., Russell, S. J., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+targeted+viral+vectors+for+gene+therapy.">Engineering targeted viral vectors for gene therapy.</a> Nat. Rev. Genet. 8, 573-587 (2007).</li><li>Magnusson, M. K., Hong, S. S., Henning, P., Boulanger, P., Lindholm, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Retargeting+of+Adenovirus+Vectors:+Functionality+of+Targeting+Ligands+and+Their+Influence+on+Virus+Viability.">Genetic Retargeting of Adenovirus Vectors: Functionality of Targeting Ligands and Their Influence on Virus Viability.</a> J. Gene Med. 4, 356-370 (2002).</li><li>Henning, P., Lundgren, E., Carlsson, M., Frykholm, K., Johannisson, J., Magnusson, M. K., TÃ¥ng, E., Franqueville, L., Hong, S. S., Lindholm, L., Boulanger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+Type+5+Fiber+Knob+Domain+has+a+Critical+Role+in+Fiber+Protein+Synthesis+and+Encapsidation.">Adenovirus Type 5 Fiber Knob Domain has a Critical Role in Fiber Protein Synthesis and Encapsidation.</a> J. Gen. Virol. 87, 3151-3160 (2006).</li><li>Zhang, M. M., Tsou, L. K., Charron, G., Raghavan, A. S., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+fluorescence+imaging+of+dynamic+S-acylation+and+protein+turnover.">Tandem fluorescence imaging of dynamic S-acylation and protein turnover.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 8627-8632 (2010).</li><li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unnatural+amino+acid+incorporation+onto+adenoviral+(Ad)+coat+proteins+facilitates+chemoselective+modification+and+retargeting+of+Ad+type+5+vectors.">Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors.</a> J. Virol. 85, 7546-7554 (2011).</li><li>Hong, V., Presolski, S. I., Ma, C., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+Optimization+of+Copper-Catalyzed+Azide-Alkyne+Cycloaddition+for+Bioconjugation.">Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation.</a> Angew. Chem. Int. Ed. 48, 9879-9883 (2009).</li><li>Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry+and+Bioorthogonal+Reactions:+Unprecedented+Selectivity+in+the+Labeling+of+Biological+Molecules.">Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules.</a> Biochemistry. 48, 6571-6584 (2009).</li><li>Agard, N. J., Prescher, J. A., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Strain-Promoted+[3+++2]+Azide-Alkyne+Cycloaddition+for+Covalent+Modification+of+Biomolecules+in+Living+Systems.">A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems.</a> J. Am. Chem. Soc. 46, 15046-15047 (2004).</li><li>Dommerholt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Readily+Accessible+Bicyclononynes+for+Bioorthogonal+Labeling+and+Three-Dimensional+Imaging+of+Living+Cells.">Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells.</a> Angew. Chem. Int. Ed. 49, 9422-9425 (2010).</li><li>Link, A. J., Vink, M. K. S., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+the+functionalizable+methionine+surrogate+azidohomoalanine+via+copper-catalyzed+diazotransfer.">Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazotransfer.</a> Nat. Protoc. 2, 1879-1883 (2007).</li></ol>

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...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>腺病毒5型（Ad5的）含有绿色荧光蛋白转基因</td><td> BCBC </td><td> 391 </td></tr><tr><td>人类胚胎肾细​​胞（HEK 293） </td><td> ATCC </td><td> CRL-1573 </td></tr><tr><td>杜尔贝科的改良Eagle培养基（DMEM培养基），高糖</td><td> Invitrogen公司</td><td> 11965-092 </td></tr><tr><td>杜尔贝科的改良Eagle培养基，无蛋氨酸，没有半胱氨酸（DMEM-蛋氨酸/半胱氨酸），高糖</td><td> Invitrogen公司</td><td> 21013-024 </td></tr><tr><td>小牛血清</td><td> Invitrogen公司</td><td> 16170-078 </td></tr><tr><td>青霉素 - 链霉素100×解决方案（笔链球菌） </td><td> Invitrogen公司</td><td> 15140-122 </td></tr><tr><td> 0.5％胰蛋白酶-EDTA（10×） </td><td&gt; Invitrogen公司</td><td> 15400-054 </td></tr><tr><td> 100 MM细胞培养皿中，组织培养处理的聚苯乙烯</td><td>屋宇署猎鹰</td><td> 353003 </td></tr><tr><td>细胞培养二氧化碳培养箱</td><td></td><td></td></tr><tr><td>血球</td><td></td><td></td></tr><tr><td>生物安全柜</td><td></td><td></td></tr><tr><td>无菌注射器过滤器（0.2微米，醋酸纤维素） </td><td>厂商VWR </td><td> 28145-477（NA）514-0061（欧洲） </td></tr><tr><td>阿凡提乙脑离心机</td><td>贝克曼</td><td> 369001 </td></tr><tr><td>锥形管（50毫升，无菌） </td><td>屋宇署猎鹰</td><td> 352098 </td></tr><tr><td>管，薄壁，超清晰，13.2毫升，14 x 89毫米</td><td>贝克曼</td><td> 344059 </td></tr><tr><td>超速离心机配备与西南41和SW 60 Rotor </td><td>贝克曼</td><td></td></tr><tr><td> Eppendorf公司Biopur安全锁管，1.5毫升</td><td> Eppendorf公司</td><td> 0030 121.589 </td></tr><tr><td>离心机5418 </td><td> Eppendorf公司</td><td> 5418 </td></tr><tr><td>离心九月凝胶过滤离心柱</td><td>普林斯顿大学分离</td><td> CS-901 </td></tr><tr><td>无菌注射针，18针</td><td></td><td></td></tr><tr><td>氮手套袋（如果进行无氧CuAAC） </td><td></td><td></td></tr><tr><td>杜瓦瓶</td><td></td><td></td></tr><tr><td>液氮</td><td></td><td></td></tr><tr><td>电泳槽</td><td></td><td></td></tr><tr><td>荧光凝胶扫描仪</td><td></td><td></td></tr><tr><td>准备凝胶的Tris-HCl凝胶</td><td> BIO-RAD </td><td> 161-1105 </td></tr><tr><td> L - Azidohomoalanine </td><td> AnaSpec </td><td> 63669 </td></tr><tr><td>耶拿生物科学</td><td> CLK-AA005 </td><td></td></tr><tr><td> peracetylated 的 N-azidoacetylgalactosamine（AC 4 GalNAz） </td><td> Invitrogen公司</td><td> C33365 </td></tr><tr><td> Thermo Scientific的</td><td> 88905 </td><td></td></tr><tr><td> Sigma-Aldrich公司</td><td> A7480 </td><td></td></tr><tr><td> bathophenanthroline二磺酸（BDA）的钠盐</td><td> MP的Biomedicals </td><td> 0215011201 </td></tr><tr><td>二甲基亚砜（DMSO） </td><td></td><td></td></tr><tr><td>甲醇</td><td></td><td></td></tr><tr><td> Tris碱（三（羟甲基）氨基甲烷） </td><td></td><td></td></tr><tr><td>磷酸氢二钠（钠2 HPO 4） </td><td></td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td></td><td></td></tr><tr><td> 1M盐酸</td><td></td><td></td></tr><tr><td>甘油</td><td></td><td></td></tr><tr><td>牛血清白蛋白</td><td></td><td></td></tr><tr><td> L-半胱氨酸</td><td></td><td></td></tr><tr><td> L-蛋氨酸</td><td></td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td></td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td></td><td></td></tr><tr><td>甘氨酸</td><td></td><td></td></tr><tr><td>溴酚蓝</td><td></td><td></td></tr><tr><td>氯化铯（CsCl） </td><td></td><td> ＆NBSP; </td></tr><tr><td>铜（I）溴化物（溴化亚铜） </td><td></td><td></td></tr><tr><td>氯化钙（ 氯化钙 ） </td><td></td><td></td></tr><tr><td>氯化钾（KCl） </td><td></td><td></td></tr><tr><td>氯化镁（MgCl 2的 ） </td><td></td><td></td></tr><tr><td>氯化钠（NaCl） </td><td></td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> 炔CuAAC *探头 </td><td></td><td></td></tr><tr><td> ：TAMRA炔</td><td> Invitrogen公司</td><td> T10183 </td></tr><tr><td colspan="3"></td></tr><tr><td> 紧张炔探头SPAAC * </td><td></td><td></td></tr><tr><td> ：TAMRA迪波炔</td><td> Invitrogen公司</td><td> C10410 </td></tr></tbody></table> *点击化学试剂和试剂盒的著名厂商包括Invitrogen公司，耶拿的生物科技公司，贝里联营公司，Sigma-Aldrich公司，格伦研究“化学工具，Baseclick。各种炔染料和靶向配体，可以发现在这些厂商的产品目录。

chemoselective modification of viral surfaces via bioorthogonal click chemistry

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。

Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

腺病毒颗粒设计，含有非天然氨基酸类似物azidohomoalanine或叠氮糖<em&gt;Ø</em&gt; 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。

通过Bioorthogonal点击化学化学选择性病毒表面改性

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Preparation of Viral DNA from Nucleocapsids

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...