Name: making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry
Uploaded: 2018-05-27T14:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

J.J.G. 承认美国德州科学基金会 (DMR-1654405) 和德克萨斯癌症预防研究所 (CPRIT) (RP170752s) 的支持。

1. 准备
<ol><li>制作溶源性汤 (磅) 琼脂和倒盘12。</li>
	<li>
用含有 wtQβ涂层蛋白序列的 pET28 质粒转化 BL21 (DE3)。
	<ol>
<li>解冻大肠杆菌BL21 (DE3) 在冰浴中的主管细胞。在离心管中放置50个细胞的 ul。</li>
		<li>将质粒的 2 ul 添加到一管中, 轻轻地轻拂管子。然后在冰上孵育30分钟。</li>
		<li>热休克细胞四十五年代在水浴那是在确切地42°c。热震后立即将管子放回冰浴中, 孵化5分钟。</li>
		<li>添加950不含任何抗生素的 LB 介质的 ul。</li>
		<li>在37摄氏度的60分钟内, 以 200 rpm 的文化摇摆。</li>
		<li>板材 100 ul 的文化在 LB 琼脂板 (与卡那霉素) 和孵化的板块隔夜在37摄氏度。需要时选择白色菌落。</li>
	</ol>
</li>
	<li>
制作超级最佳的汤 (呜咽) 介质。
	<ol>
<li>蒸压釜两个2升困惑锥形烧瓶 superdry 循环。</li>
		<li>在无菌环境中, 称量并添加20.0 克胰蛋白胨, 5.0 克酵母萃取物, 2.469 克无水硫酸镁, 0.584 克氯化钠和0.186 克氯化钾到每瓶。</li>
		<li>把体积在每瓶和高压釜的液体循环中的超纯水1升。</li>
		<li>一旦呜咽的媒体已经达到室温后热处理, 添加1毫升卡那霉素 (100 毫克/毫升) 每公升的媒体和存储在4摄氏度。</li>
	</ol>
</li>
	<li>
使0.1 米磷酸钾缓冲液 (pH 7.00)。
	<ol>
<li>在500毫升的超纯水中溶解68.045 克磷酸钙钾, 使1米磷酸钙溶液。</li>
		<li>在500毫升超纯水中溶解87.09 克钾二元, 使二元钾溶液1米。</li>
		<li>将38.5 毫升的钾磷酸钙溶液和61.5 毫升的二元钾添加到1升瓶中。</li>
		<li>如果需要, 将 pH 值调整为 7.00, 并使其体积为1升。</li>
	</ol>
</li>
	<li>
在0.1 米磷酸钾缓冲液中5–40% 蔗糖梯度 (pH 值为 7.00)。
	<ol>
<li>在50毫升离心管中, 用 5–40% (增加 5%) 蔗糖溶解于0.1 米磷酸钾缓冲液 (pH 7.00) 制备溶液。</li>
		<li>用一根长针注射器在38毫升的聚碳酸酯管底部沉积3.3 毫升5% 蔗糖溶液, 并对五其他管进行重复。</li>
		<li>小心地将3.3 毫升的10% 蔗糖溶液放在管子的底部, 小心地除去针, 以免扰乱渐变。重复其他五管。 
		 </li>
	</ol>
</li>
	<li>继续存入3.3 毫升的蔗糖溶液层, 每管增加15% 到 40%, 同时小心不干扰梯度。</li>
	<li>完成后, 用铝箔覆盖渐变的顶部, 并存储在-80 摄氏度。</li>
</ol>2. Qβ的表达
<ol><li>用1:1 漂白剂/乙醇擦拭工作台面积。</li>
	<li>在无菌环境中, 通过将大肠杆菌BL21 (DE3) 的单个菌落添加到3毫升的无菌环境中, 使两个3毫升的起始培养物处于不细菌的氛围中。</li>
	<li>在一个 250 rpm 在37摄氏度和0% 相对湿度 (rH) 房间一夜之间增长。</li>
	<li>
在呜咽媒体中接种起始文化:
	<ol>
<li>从振动筛中取走3毫升的起始培养物, 在无菌环境中, 将每种起始文化倒入两个 2 l 困惑的锥形烧瓶中的一个, 每瓶有1升的新鲜呜咽介质。</li>
		<li>将接种的介质放在 250 rpm 的振动筛上, 在37摄氏度和0% 个 rH 室。</li>
	</ol>
</li>
	<li>在37摄氏度和 0% rH 室中, 在 250 rpm 的振动筛上种植细菌, 直到 OD600到达0.9–1.0。</li>
	<li>用 P1000 吸管添加1毫升1米异丙基β-d-1 thiogalactopyranoside (IPTG), 诱导蛋白表达。</li>
	<li>在37摄氏度和 0% rH 房间过夜时, 将媒体放在 250 rpm 的振动筛上。</li>
	<li>第二天早上从振动筛中取出介质, 用1000毫升的瓶子在 20621 x 克上用1小时在4摄氏度处收割细胞。</li>
	<li>
丢弃上清液并收集细胞颗粒。
	<ol>
<li>将上清液倒入一瓶约5毫升漂白剂, 以杀灭细菌。这是浪费。</li>
		<li>使用铲刮从离心瓶底部的细胞颗粒, 并把球团成50毫升离心管。</li>
	</ol>
</li>
</ol>3. Qβ的纯化
<ol><li>并用重悬20–30毫升0.1 米磷酸钾缓冲细胞颗粒 (pH 7.00)。</li>
	<li>请确保泥沙没有块, 并根据制造商的协议溶解单元格使用 microfluidizer 处理器 (请参见材料表)。溶解细胞至少两次, 以增加粒子的产量。</li>
	<li>离心250毫升离心机瓶在 20621 x g 1 小时在4摄氏度的裂解。</li>
	<li>丢弃颗粒, 测量 mL 中清液的体积。将该值乘以 0.265, 并将硫酸铵的 g 量加到上清。</li>
	<li>在 200 rpm 的搅拌板上搅动4摄氏度, 至少1小时, 以沉淀出蛋白质。</li>
	<li>离心机在250毫升瓶在 20621 x g 1 小时在4°c。</li>
	<li>用大约10毫升的0.1 米磷酸钾缓冲液 (pH 7.00) 丢弃上清和并用重悬颗粒。</li>
	<li>将等量的1:1 氯仿/正丁醇添加到粗样品中, 用涡流混合几秒钟。</li>
	<li>离心机在38毫升管在 20621 x g 30 分钟在4°c。</li>
	<li>使用巴斯德吸管回收顶部水层。小心不要采取在水和有机层之间形成的凝胶状层。</li>
	<li>解冻六5–40% 预先做的蔗糖梯度和装载大约2毫升提取物到每个。</li>
	<li>Ultracentrifuge 在 99582 x g 为16小时在4°c 与自由减速。</li>
	<li>发光二极管 (LED) 光在每个管下, 蓝带应该变得可见。用长针注射器回收这些微粒。</li>
	<li>Ultrapellet 的粒子在 370541 x g 2.5 小时在4摄氏度。</li>
	<li>用0.1 米磷酸钾缓冲液 (pH 为 7.00), 丢弃上清和并用重悬纯化颗粒的透明颗粒。</li>
</ol>4. 产品的数量和确认
<ol><li>使用布拉德福德检测来量化产品13。</li>
	<li>运行还原和不还原的月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 确认产品14。 注: 减少 SDS 页用于确定涂层蛋白的分子量;不缩减的 SDS 页面是确认高阶结构。</li>
</ol>5. Qβ上的共轭 DB 化合物
<ol><li>
减少 Qβ上的二硫化物。
	<ol>
<li>将0.0020 克的三 (2-羧乙基) 膦 (TCEP) 溶解成1毫升的超纯水, 以制造100x 的库存溶液。 注: 在还原前准备新鲜 TCEP。</li>
		<li>添加200µL 的 Qβ (5 毫克/毫升) 到离心管。</li>
		<li>通过添加20µL 100x TCEP 库存解决方案进行跟踪。</li>
		<li>室温孵育1小时。</li>
	</ol>
</li>
	<li>
用 dibromomaleimide 聚乙二醇 (DB PEG) 重新桥降二硫醚。
	<ol>
<li>在100µL 的 DMF 中溶解0.0017 克 dibromomaleimide 聚乙二醇 (DB PEG)。</li>
		<li>添加680µL 10 毫米磷酸磷酸钠溶液 (pH 5.00)。</li>
		<li>将减少的 Qβ加入到 DB 的溶液中, 观察 365 nm 紫外线灯下的混合过程。明亮的黄色荧光可以立即可视与365纳米手持紫外线灯在混合。</li>
		<li>让反应在室温下 (RT) 在烤上一夜之间进行。</li>
	</ol>
</li>
	<li>用离心过滤器 (COMW = 10 kDa) 净化反应混合物三次使用 1x PBS 在 3283 x g 20 分钟在4°c。</li>
	<li>通过非还原 SDS 页和本机琼脂糖凝胶电泳对共轭进行监测。 注: VLPs 在本机琼脂糖凝胶中作为完整的颗粒运行, 并根据其电荷、大小和形状分离。</li>
</ol>

<ol>
	<li>Pokorski, J., Breitenkamp, K., Liepold, L., Qazi, S., 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=Functional+Virus-Based+Polymer-Protein+Nanoparticles+by+Atom+Transfer+Radical+Polymerization.">Functional Virus-Based Polymer-Protein Nanoparticles by Atom Transfer Radical Polymerization.</a> J. Am. Chem. Soc. 133 (24), 9242-9245 (2011).</li><li>Capehart, S., Coylet, M., Glasgow, J., Francis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Integration+of+Gold+Nanoparticles+and+Organic+Fluorophores+Using+Synthetically+Modified+MS2+Viral+Capsids.">Controlled Integration of Gold Nanoparticles and Organic Fluorophores Using Synthetically Modified MS2 Viral Capsids.</a> J. Am. Chem. Soc. 135 (8), 3011-3016 (2013).</li><li>Li, S., 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=Template-Directed+Synthesis+of+Porous+and+Protective+Core-Shell+Bionanoparticles.">Template-Directed Synthesis of Porous and Protective Core-Shell Bionanoparticles.</a> Angew. Chem. Int. Ed. 55 (36), 10691-10696 (2016).</li><li>Chen, Z., Li, N., Li, S., Dharmarwardana, M., Schlimme, A., Gassensmith, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Chemistry:+The+Chemical+Functionalization+of+Viral+Architectures+to+Create+New+Technology.">Viral Chemistry: The Chemical Functionalization of Viral Architectures to Create New Technology.</a> WIREs. Nanomed. Nanobiotechnol. 8 (4), 512-534 (2015).</li><li>Chalker, J. M., Bernardes, G. J. L., Lin, Y. A., Davis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+proteins+at+cysteine:+opportunities+in+chemistry+and+biology.">Chemical modification of proteins at cysteine: opportunities in chemistry and biology.</a> Chem. - Asian J. 4 (5), 630-640 (2009).</li><li>Le, D. H., Lee, K. L., Shukla, S., Commandeur, U., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+Virus+X,+a+Filamentous+Plant+Viral+Nanoparticle+for+Doxorubicin+Delivery+in+Cancer+Therapy.">Potato Virus X, a Filamentous Plant Viral Nanoparticle for Doxorubicin Delivery in Cancer Therapy.</a> Nanoscale. 9 (6), 2348-2357 (2017).</li><li>Chen, L., Wu, Y., Yuan, L., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-templated+FRET+Platform+for+the+Rational+Design+of+Ratiometric+Fluorescent+Nanosensors.">Virus-templated FRET Platform for the Rational Design of Ratiometric Fluorescent Nanosensors.</a> Chem. Comm. 51 (50), 10190-10193 (2015).</li><li>Lee, P., 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=Polymer+Structure+and+Conformation+Alter+the+Antigenicity+of+Virus-like+Particle-Polymer+Conjugates.">Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle-Polymer Conjugates.</a> J. Am. Chem. Soc. 139 (9), 3312-3315 (2017).</li><li>Zhang, X., 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=Polymer-Protein+Core-Shell+Nanoparticles+for+Enhanced+Antigen+Immunogenicity.">Polymer-Protein Core-Shell Nanoparticles for Enhanced Antigen Immunogenicity.</a> ACS Macro Lett. 6 (4), 442-446 (2017).</li><li>Brown, S. D., Fielder, J. D., 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=Assembly+of+Hybrid+Bacteriophage+Qbeta+virus-like+particles.">Assembly of Hybrid Bacteriophage Qbeta virus-like particles.</a> Biochemistry. 48 (47), 11155-11157 (2009).</li><li>Chen, Z., 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=Fluorescent+Functionalization+across+Quaternary+Structure+in+a+Virus-+like+Particle.">Fluorescent Functionalization across Quaternary Structure in a Virus- like Particle.</a> Bioconjugate Chem. 28 (9), 2277-2283 (2017).</li><li>. Pouring LB Agar Plates Available from: <a target="_blank" href="https://www.addgene.org/protocols/pouring-lb-agar-plates/">https://www.addgene.org/protocols/pouring-lb-agar-plates/</a> (2016)</li><li>Smith, M., 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=Protein+Modification,+Bioconjugation,+and+Disulfide+Bridging+Using+Bromomaleimides.">Protein Modification, Bioconjugation, and Disulfide Bridging Using Bromomaleimides.</a> J. Am. Chem. Soc. 132 (6), 1960-1965 (2010).</li><li>Castaneda, L., 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=A+Mild+Synthesis+of+N-functionalised+Bromomaleimides,+Thiomaleimides+and+Bromopyridazinediones.">A Mild Synthesis of N-functionalised Bromomaleimides, Thiomaleimides and Bromopyridazinediones.</a> Tetrahedron Lett. 54 (27), 3493-3495 (2013).</li><li>Fiedler, J., 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=Engineered+Mutations+Change+the+Structure+and+Stability+of+a+Virus-+Like+Particle.">Engineered Mutations Change the Structure and Stability of a Virus- Like Particle.</a> Biomacromolecules. 13 (8), 2339-2348 (2012).</li><li>Manzenrieder, F., Luxenhofer, R., Retzlaff, M., Jordan, R., 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=Stabilization+of+Virus-like+Particles+with+Poly(2-oxazoline)s.">Stabilization of Virus-like Particles with Poly(2-oxazoline)s.</a> Angew. Chem. Int. Ed. 50 (11), 2601-2605 (2011).</li><li>Chen, Z., Li, N., Chen, L., Lee, J., Gassensmith, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Functionalized+Bacteriophage+Q&#946;+as+a+Photocaged+Drug+Carrier.">Dual Functionalized Bacteriophage Q&#946; as a Photocaged Drug Carrier.</a> Small. 12 (33), 4563-4571 (2016).</li></ol>

作者声明他们没有竞争的财政利益。

与较小的蛋白质纯化相比, 纯化噬菌体 Qβ的一个独特步骤是蔗糖梯度离心。在氯仿/正丁醇萃取步骤后, 用5-40% 蔗糖梯度进一步纯化 Qβ。在离心过程中, 粒子根据它们的大小分开。更大的微粒旅行到更高的密度区域, 而小微粒停留在低密度区域。Qβ移动到梯度的下第三, 并保持在那里, 而较小的蛋白质杂质被困在离心管的顶部。Qβ的胶体悬浮在蔗糖梯度中表现出强烈的廷德尔散射, 当 LED 光从下面照射出来时, 它可以被视为蓝色的波段。这个波段, 然后容易提取使用长注射器针。Qβ在蔗糖被离心, 产生一个明确的颗粒。该颗粒可悬浮到所需的缓冲液中, 或通过快速蛋白液体色谱 (FPLC) 进一步纯化。结果粒子的纯度可以通过 SDS 页来确定。
TCEP 在磷酸盐缓冲液中不稳定, 因此在还原前应在水中新制备 100x TCEP 溶液。此外, TCEP 既不含游离硫醇, 也不对其他氨基酸作出反应, 因此在进行共轭反应之前没有必要去除过量的 TCEP。Dibromomaleimide 化合物在 pH 值5.00 磷酸钠溶液中溶解, 随后加入还原 Qβ。在 pH 值 5.00, 半胱氨酸 (还原二硫化物) 是质子, 通过防止再氧化回二硫化物, 促进共轭反应与 dibromomaleimide。在365纳米紫外线照射下, 反应混合物在 Qβ加入 DB 化合物后立即发出黄色荧光。与附加预合成显影在 Qβ上不同, 这种荧光是由共轭反应引起的。荧光在硫磺和 maleimide 圆环之间的新的纽带被创造以后形成。荧光光谱显示, 激发 (400 nm) 和发射 (550 nm) 最大值适合在共焦显微镜中设置的紫外荧光蛋白过滤器 (λex = 405 nm, λem = 500−540 nm)。Qβ-PEG, 然后孵化与巨噬细胞生264.7 在无血清 DMEM。经过四小时的孵化, Qβ小白菜植株吸, 有斑点的黄色荧光隔间可以在细胞内可视化。需要提及的一个事实是, 荧光可以在一定程度上转移到血清中的牛血清白蛋白 (BSA) 或溶酶体内或晚期内涵体的其他半胱氨酸丰富物质中。随着时间的推移, 这将削弱细胞内的荧光细胞跟踪应用;然而, 它也为药物输送系统的新设计提供了机会。
最后, 通过 dibromomaleimide-硫醇化学, 我们在 Qβ vip 上展示了共轭 DB。functionalizes 的共轭反应不仅使二硫化物沿孔在 vip 与 PEG, 但也成为荧光标记蛋白质纳米粒子。

使用纳米病毒衣壳已成为一个令人兴奋的领域, 目的是扩大在生物医学研究领域的应用范围 1, 2, 3.Recombinantly 表达的病毒样粒子 (VLPs) 的结构来源于病毒, 但它们缺乏原始的病毒遗传物质, 使得它们无传染性的蛋白质纳米粒子。由于表面特征是基因编制的, 并且每个衣壳在它之前和之后被表达与它是相同的, 有可能知道氨基酸的无功侧链的位置和数量以原子精确度。在许多情况下, 外部和内部表面都具有多种溶剂暴露的氨基酸残留物, 这可以通过 bioconjugation 反应进行功能化--在分子和合成物之间形成共价键的反应。分子4,5。
Bioconjugation 反应帮助生物分子在相对简单的方式有更多不同的功能。感兴趣的分子, 如治疗药物6, 荧光标记7和聚合物8,9可以预先合成和特征, 然后它们被附着在 VLPs 表面上。在生物医学和生物材料研究中, 一个特别常见的 vip 是基于噬菌体 Qβ的 vip, 正如 recombinantly 所表达的, 它是一个28纳米 icosahedral 病毒衣壳10。Qβ上最常见的反应部位是 lysines, 尽管最近我们已经将 dibromomaleimide 化合物的成功共轭11与二硫化物, 通过 Qβ-贝克反应将 Haddleton 的孔隙线进行了传递。反应的收益率很好, 同样重要的是, 不会失去粒子的热稳定性。同时, 这种反应产生共轭诱导荧光, 可用于跟踪这些微粒对细胞的吸收。在这项工作中, 我们展示了聚乙二醇 (PEG) 在 Qβ表面上的共轭, 通过 Haddleton 反应, 这导致了一个明亮的黄色荧光。这些粒子可以被跟踪, 因为它们被细胞所接受。本协议将帮助研究人员生成基于 Qβ的新型荧光 PEGylated 蛋白质纳米粒子, 尽管它的原理适用于含有溶剂暴露二硫化物的许多其它 VLPs 中的一种。

<table><tbody><tr><td>LB Broth (Miller)&amp;nbsp;</td><td>EMD Millipore</td><td>1.10285.0500</td><td></td></tr><tr><td>Tryptone, Poweder</td><td>Research Products International</td><td>T60060-1000.0</td><td></td></tr><tr><td>Yeast Extract, Poweder</td><td>Research Products International</td><td>Y20020-1000.0</td><td></td></tr><tr><td>Anhydrous magnesium sulfate</td><td>P212121</td><td>CI-06808-1KG</td><td></td></tr><tr><td>Sodium Chloride (Crystalline/Certified ACS)</td><td>Fisher Scientific</td><td>S271-10</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>Elga PURELAB Flex 3 Water Purification System</td><td>Fisher Scientific</td><td>4474524</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Fisher Scientific</td><td>BP362-1</td><td></td></tr><tr><td>Potassium Phosphate Dibasic Anhydrous</td><td>Fisher Scientific</td><td>P288-500</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S25590B</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma Aldrich</td><td>I6758-1G</td><td></td></tr><tr><td>Fiberlite F10-4x1000 LEX rotor&amp;nbsp;</td><td>Fisher Scientific</td><td>096-041053</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>P212121</td><td>KW-0066-5KG</td><td></td></tr><tr><td>Chloroform</td><td>Alfa Aesar</td><td>32614-M6</td><td></td></tr><tr><td>1-Butanol</td><td>Fisher Scientific</td><td>A399-4</td><td></td></tr><tr><td>SW 28 Ti Rotor, Swinging Bucket, Aluminum</td><td>Beckman Coulter</td><td>342204: SW 28 Ti Rotor/ 342217: Bucket Set</td><td></td></tr><tr><td>Type 70 Ti Rotor, Fixed Angle, Titanium, 8 x 39 mL,</td><td>Beckman Coulter</td><td>337922</td><td></td></tr><tr><td>Coomassie (Bradford) Protein Assay</td><td>Fisher Scientific</td><td>PI23200</td><td></td></tr><tr><td>TRIS Hydrochloride</td><td>Research Products International</td><td>T60050-1000.0</td><td></td></tr><tr><td>Tetramethylethylenediamine</td><td>Alfa Aesar</td><td>J63734-AC</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma Aldrich</td><td>C4706-2G</td><td></td></tr><tr><td>2 3-Dibromomaleimide 97%</td><td>Sigma Aldrich</td><td>553603-5G</td><td></td></tr><tr><td>Polythylene Glycol</td><td>Alfa Aesar</td><td>41561-22</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Fisher Scientific</td><td>AC424375000</td><td></td></tr><tr><td>Acrylamide/bis-Acrylamide</td><td>P212121</td><td>RP-A11310-500.0</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L3771-100G</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Fisher Scientific</td><td>BP179-100</td><td></td></tr><tr><td>FV3000 confocal laser scanning microscope</td><td>Olympus&amp;nbsp;</td><td>FV3000&amp;nbsp;</td><td></td></tr><tr><td>Labnet Revolver Adjustable Rotator&amp;nbsp;</td><td>Thomas Scientific&amp;nbsp;</td><td>1190P25&amp;nbsp;</td><td></td></tr><tr><td>1000 mL Sorvall High Performance Bottle, PC, with Aluminum Cap&amp;nbsp;</td><td>Thermo Scientific</td><td>010-1459</td><td></td></tr><tr><td>Nalgene Centrifuge Bottles with Caps, Polypropylene Copolymer</td><td>Thermo Scientific</td><td>3141-0250</td><td></td></tr><tr><td>Nunc Round-bottom tubes; 38 mL; PC</td><td>Thermo Scientific</td><td>3117-0380</td><td></td></tr><tr><td>2 L Narrow Mouth Erlenmeyer Flasks with Heavy Duty Rim</td><td>Pyrex</td><td>4980-2L</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Units</td><td>Millipore Sigma</td><td>UFC801024</td><td></td></tr><tr><td>M-110P Microfluidizer Materials Processor</td><td>Microfluidics</td><td>M-110P</td><td></td></tr><tr><td>Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes</td><td>Thermo Scientific</td><td>3117-0380PK</td><td></td></tr><tr><td>Bottle, with Cap Assembly, Polycarbonate</td><td>Beckman Coulter</td><td>41121703</td><td></td></tr><tr><td>Cylinder, Graduated - Polypropylene 250 mL</td><td>PolyLab</td><td>80005</td><td></td></tr><tr><td>533LS-E Series Steam Sterilizers</td><td>Getinge</td><td>533LS-E</td><td></td></tr><tr><td>TrueLine, Cell Culture Plate, Treated, PS, 96 Well, with Lid</td><td>LabSource</td><td>D36-313-CS</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tube</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>Microcentifuge Tube: 1.5mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>VWR Os-500 Orbital Shaker</td><td>VWR Scientifc Products</td><td>14005-830</td><td></td></tr><tr><td>Tetra Handcast systems</td><td>Bio-Rad</td><td>1658000FC</td><td></td></tr><tr><td>Polypropylene, 250 mL</td><td>Beckman Coulter</td><td>41121703</td><td></td></tr><tr><td>Spectrofluorometer NanoDrop</td><td>Thermo Fisher Scientific</td><td>3300</td><td></td></tr><tr><td>Long Needle&amp;nbsp;</td><td>Hamilton&amp;nbsp;</td><td>7693</td><td></td></tr><tr><td>Exel International 5 to 6 cc Syringes Luer Lock</td><td>Fisher Scientific</td><td>14-841-46</td><td></td></tr><tr><td>P1000 Pipetman</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>P200 Pipetman</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>P100 Pipetman</td><td>Gilson</td><td>F123615</td><td></td></tr><tr><td>P20 Pipetman</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>P10 Pipetman</td><td>Gilson</td><td>F144802</td><td></td></tr><tr><td>Intel Weighing PM-100 Laboratory Classic High Precision Laboratory Balance</td><td>Intelligent Weighting Technology</td><td>IWT_PM100</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tube</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>4&amp;ndash;15% Mini-PROTEAN TGX Gel, 10 well, 50 &amp;micro;l</td><td>Bio-Rad</td><td>456-1084</td><td></td></tr></tbody></table>

making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry

最近在生物医学和材料研究中出现的病毒样粒子 (VLPs) 的增加, 可以归因于它们的生物合成、离散大小、遗传可编程性和生物降解能力的简化。虽然他们很容易 bioconjugation 反应, 添加合成配体在其表面上, bioconjugation 方法的范围, 这些水中出生的衣壳是相对有限的。为了促进功能性生物材料研究的方向, 必须考虑非传统的 bioconjugation 反应。本协议中描述的反应使用 dibromomaleimides 在噬菌体 Qβ的基础上引入了一个 vip 的溶剂暴露二硫化物键的新功能。此外, 最终产品是荧光, 它具有生成追踪体外探针使用商业上可用的筛选器集的额外好处。

dibromomaleimide 衍生物可通过 dibromomaleimide 酸酐与主胺15之间的缩合反应合成。另外, 使用 n-羧酸激活 34-dibromomaleimide 的温和合成方法16在这里被开发了与 methoxypolyethylene 乙二醇 (peg) 反应产生 DB peg (图 1)。核磁共振用于识别复合结构 (图 2)。Qβ vip 是一个28纳米 icosahedral 蛋白质纳米粒子, 这是由180相同的涂层蛋白组成。外套蛋白质倾向于形成 noncovalent 联锁脂肪酸通过他们的α螺旋领域与β板料从毗邻外套蛋白质17。VLPs 倾向于在高温、极端小灵通和各种溶剂组合物18中的稳定性选择。在这种情况下, Qβ vip 比其他 RNA 噬菌体在Leviviridac家族中更稳定, 原因是180股之间的二硫化物债券位于五和六倍的对称轴上的外壳,19 (图 3)。这些 hexameric 和 pentameric 结构由二硫化物链接, 可通过非缩减的 SDS 19 来可视化.十当量的 TCEP (三 (2-羧乙基) 膦) (0.70 µmol), 相对于二硫化物在1毫克 Qβ (0.070 µmol 涂层蛋白, 0.070 µmol 二硫化物), 用于减少所有二硫化物产生减少 Qβ衣壳 (rQβ) 在室温下, 在一个小时和非缩减的 SDS 页面显示, 所有的高阶结构都减少到单体涂层蛋白 (图 4 和图 5A)。rebridge 的反应是作为一锅合成, 因为粗 rQβ混合料直接添加到20当量的解决方案, 在磷酸钠 (10 毫米, pH 5.00, 10% DMF) 后, 一小时的还原反应11。有可观测的明亮的黄色荧光在365毫微米紫外灯之下 (图 5D), 在加法以后紧接着 DB PEG。该混合物然后孵化在 RT 过夜的烤, 其次是净化使用离心过滤器 (截留分子量 = 10 kDa) 冲洗与所需的缓冲三倍, 以消除过剩的数量的小分子。Qβ-malemide 共轭悬浮成10毫米磷酸钠溶液 (pH 5.00) 以促进钴。在紫外和考马斯蓝色染色 (图 5A) 下, 不还原的 SDS 页确认了共轭。所有的波段显示荧光 (图 5A) 在紫外线下 colocalized 与考马斯蓝色染色, 代表一个成功的共轭。用本机琼脂糖凝胶电泳和透射电镜 (TEM) (图 5B、C) 证实了 Qβ PEG 共轭物的完整性。
荧光光谱显示了 Qβ-maleimide (Qβ) 和 Qβ PEG 的激发和发射最大值分别约为 400 nm 和 540–550 nm (图 6)。这与商业上可利用的 GFP-紫外线过滤器集合, 励磁波长是405毫微米和发射波长是500–540毫微米。将共轭物的物理性质与商用过滤器集方便地对齐, 允许使用 Qβ作为体外探针, 该探测器在图 7中完成并成像。Qβ (200 nM) 在无血清 DMEM 培养基中与小鼠 Raw-264.7 细胞一起孵化, 其次为细胞核染色。图 7中的定位图像显示, 黄色荧光粒子被 Raw-264.7 细胞小白菜植株吸, 在孵化四小时后可以跟踪。unfunctionalized Qβ VLPs 显示可忽略的荧光。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57712/57712fig1.jpg"> 图 1: DB 的合成方案.<a href="/files/ftp_upload/57712/57712fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57712/57712fig2.jpg"> 图 2: 核磁共振特性.(A)1H 核磁共振和(B) 13C 的 DB PEG。<a href="/files/ftp_upload/57712/57712fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57712/57712fig3.jpg"> 图 3: 嵌合体中处理的 Qβ vip 衣壳的晶体结构 (PDB ID: 1QBE).两个半胱氨酸残留物 (胱氨酸74和胱氨酸 80) 呈橙色。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57712/57712fig4.jpg"> 图 4: Qβ-maleimide (Qβ) 共轭结合方案.Qβ (0.07 µmol 二硫化物) 减少使用10当量的 TCEP (0.7 µmol) 在 RT 一个小时, 然后增加20当量 dibromomaleimide 化合物 (db 和 db PEG) (1.4 µmol)。<a href="/files/ftp_upload/57712/57712fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57712/57712fig5.jpg"> 图 5: Qβ、rQβ、Qβ和 Qβ钉的特性.(A)不减少的 SDS 页和(B)本机琼脂糖凝胶的 Qβ, rQβ, Qβ米和 Qβ PEG 下紫外 (顶部) 和考马斯蓝染色 (底部)。(C) Qβ (顶部) 和 Qβ钉 (底部) 的 TEM 显微图像。(D) Qβ-PEG 反应混合物在365纳米紫外线照射下的照片。<a href="/files/ftp_upload/57712/57712fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57712/57712fig6.jpg"> 图 6: 荧光光谱.荧光激发(A)和发射(B)光谱的 Qβ和 Qβ-PEG 在0.1 米磷酸钾缓冲 (pH 值 7.00)。励磁最大值约为 400 nm, 发射最大值在 540–550 nm 附近。<a href="/files/ftp_upload/57712/57712fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57712/57712fig7.jpg"> 图 7: 巨噬细胞264.7 单元中 Qβ-PEG 共轭的共焦荧光图像.蓝色: NucRed 活 647 ReadyProbes 试剂。黄色: Qβ。顶部图像: 合并的蓝色和黄色通道。底部图像: 明亮的字段图像。过滤器集: 紫外荧光蛋白 (λex = 405 毫微米, λem = 500–540 nm), Cy5。孵化时间为4小时.<a href="/files/ftp_upload/57712/57712fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Dibromomaleimide 二硫化学制备共轭诱导荧光 PEGylated 病毒样粒子

在这里, 我们提出一个程序, 荧光 functionalize 二硫化物 Qβ vip 与 dibromomaleimide。我们描述了 Qβ的表达和纯化, dibromomaleimide 功能分子的合成, dibromomaleimide 和 Qβ的共轭反应。生成的黄色荧光共轭粒子可作为细胞内的荧光探针。

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

making-conjugation-induced-fluorescent-pegylated-virus-like-particles

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.8K 次观看.  University of Texas at Dallas. 在这里, 我们提出一个程序, 荧光 functionalize 二硫化物 Qβ vip 与 dibromomaleimide。我们描述了 Qβ的表达和纯化, dibromomaleimide 功能分子的合成, dibromomaleimide 和 Qβ的共轭反应。生成的黄色荧光共轭粒子可作为细胞内的荧光探针。

视频: Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

科研

免疫与感染

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

生物工程

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

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 ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

生物学

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

重组呼吸道合胞病毒的生成、扩增和滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

Dibromomaleimide 二硫化学制备共轭诱导荧光 PEGylated 病毒样粒子

摘要

探索更多视频

此视频中的章节