提交人承认, 资助 RHC 由 NIH 赠款 # 的 AI095382, EB021230, CA198880, 国家粮食和农业研究所, 以及芬兰杰出的教授计划。

1. 昆虫细胞的 HEVNP 生产
注意: 所有以下步骤都应在单元格培养罩中执行。有关更详细的 HEVNP 生产过程11, 请参阅我们以前的出版物。
<ol><li>在昆虫细胞介质中培养 Sf9 细胞 (参见材料表), 以 50-75% 汇流在6井板上。</li>
	<li>利用昆虫细胞转染试剂根据制造商的协议, 染 Bacmids 含有 HEVNP-573C ORF2 到 Sf99 细胞生产重组杆状病毒。根据细胞的生存能力, 在27摄氏度孵育转染细胞, 3-6 天。</li>
	<li>在感染后 3-6 天收集上清液 (转染后的细胞因杆状病毒感染裂解) 为 P0 杆状病毒。</li>
	<li>在25厘米2单层烧瓶中应用200µL P0 库存到 50-75% 汇合 Sf9 细胞之前, 移除培养基。每15分钟摇动烧瓶, 确保接种的全部覆盖。重复4次, 总共60分钟。</li>
	<li>添加2毫升的昆虫细胞培养培养基到烧瓶, 并保持在27摄氏度 3-6 天, 取决于细胞的生存能力, 放大杆状病毒, 以更高的效价。</li>
	<li>进行斑块化验以获得杆状病毒滴度阅读12。</li>
	<li>培养悬浮的 Tn5 昆虫细胞 (材料表) 与100毫升昆虫细胞媒介在250毫升瓶并且在27°c 在150转到一个滴度 0.5 x 105 -1 x 106为接种。</li>
	<li>添加杆状病毒在 5-10 到100毫升 Tn5 细胞在250毫升瓶中的多重感染 (语言)。接种后, 在27°c 以150转每分钟晃动 5-7 天。</li>
	<li>一旦大多数细胞出现囊泡, 70-90% 的细胞在光学显微镜观察下死亡, 收集 Tn5 细胞并转移到33毫升 ultracentrifuge 管。将 ultracentrifuge 管放在摆动斗转子中, 平衡, 并将细胞碎片和重组杆状在 1万 x g 处旋转90分钟, 达到25摄氏度。</li>
	<li>将含有释放 HEVNPs 的上清液保持在4摄氏度, 以进一步纯化。为进一步贮存杆状病毒上清, 添加蛋白酶抑制剂。</li>
</ol>2. HEVNP 纯化
<ol><li>用氯化铯 (中海) 梯度分离 HEVNPs 颗粒和分离:<ol>
<li>转移收集到的上清 (从步骤 1.10), 并添加 20% NP-40 到每个管, 使最终浓度 0.5% NP-40, 以溶解任何剩余的细胞膜。轻轻地混合吹打和孵化至少30分钟在25摄氏度。</li>
		<li>Ultracentrifuge HEVNPs 在 112400 x g 在摆动的桶式转子为2小时在4°c 从上清的 HEVNPs 颗粒下来。离心后丢弃上清, 在每管200µL 10 毫米的 MES 缓冲 pH 值 6.2 (O/N) 在4摄氏度的情况下, 轻轻地重新悬浮原油颗粒。运行 SDS 页 (步骤 3.1) 以确认 ORF2 在粗颗粒中存在 HEVNP, 作为 52 kDa 带。</li>
		<li>在6.2 毫升 ultracentrifuge 管中混合1.96 克中海, 再悬浮的粗球团, 4 毫升0.01 米的 MES pH 5, 准备 38.5% (w/v) 中海梯度。平衡管和地方在摆动斗转子。Ultracentrifuge 在 14.7万 x g 16 小时在4摄氏度。</li>
	</ol>
</li>
	<li>收集了中海梯度后的分数:<ol>
<li>丢弃前500µL 分数, 这主要是轻量细胞膜碎片。收集500µL 分数, 从管的顶端开始, 并改变每个分数之间的提示。将每个分数放入编号/标记为1.5 毫升的管。 注: 由于中海中高浓度, HEVNP ORF2 的存在, 不能通过运行 SDS 页凝胶来检测。中海中的每一个分数可以被删除或稀释通过遵循中海清洁程序。另外, 中海梯度可由 10-40% 蔗糖梯度13取代, 以避免中海中残留。</li>
		<li>把每一个分数转移到一个5毫升 ultracentrifuge 管和稀释中海中与4.5 毫升10毫米 MES, pH 值6.2。平衡管和地方在摆动斗转子。Ultracentrifuge 在 14.7万 x g 2 小时, 在4°c, 以球团下来的 HEVNPs。</li>
		<li>放弃上清, 并轻轻地重新悬浮在100µL 10 毫米 MES pH 6.2 在每管 HEVNPs。覆盖管, 以避免蒸发和孵化4摄氏度的 O/N。</li>
		<li>使用分光光度计记录 A280 读数和 A260/A280 nm 比值。确定 ORF2 的近似浓度为: <img alt="Equation" src="/files/ftp_upload/57020/57020eq1.jpg"> 每个 ORF2 将包含1胱氨酸站点和1赖氨酸站点为化学共轭。 注: HEVNP ORF2 的摩尔消光系数为 60280, 相当于1.019 毫克/毫升 x A280。这是如此接近 1:1, HEVNPs 的浓度 (在镁/毫升) 可以接近的 A280, 因此, ORF2 的浓度由上述方程式。例如, A280 读数为1的 HEVNP 将有1毫克/毫升的浓度, 相当于18.8 µM ORF2。</li>
		<li>准备一个 SDS 页面。使用每个分数的6µL 样本来确定含有 53.3 kDa HEVNP ORF2 蛋白的分数 (步骤 3.1)。HEVNPs 应该被发现在分数 3-5, 与密度为 ~ 1.25 克/毫升。</li>
		<li>通过 TEM 观察, 确定 HEVNPs 的存在和纯度。准备或稀释 HEVNP 样品到 0.5-2.0 毫克/毫升的 TEM。HEVNPs 以透射电镜显示为空 icosahedral 蛋白, 直径约 27 nm (图 2)。一些蛋白质污染物可能留在分数, 并将在 TEM 下观察 (步骤 3.2)。</li>
	</ol>
</li>
	<li>在透射电镜下存在杂质的情况下, 重复步骤 2.1.3 2.2.6, 以提高 HEVNPs 的纯度。 注: 通过中海梯度的额外净化可能导致 HEVNPs 的产量损失。另外, 中海梯度纯化可以用 10-40% 蔗糖梯度代替, 以避免中海中的残留13。</li>
</ol>3. HEVNP 特征
<ol><li>准备 SDS 页4-12% 双三蛋白凝胶, 1.0 毫米, 17 井 (见材料表) 根据用户手册14:<ol>
<li>添加2µL 4x 加载缓冲到6µL 蛋白质样品。将样品混合物在热块中孵育10分钟, 在100摄氏度变性蛋白质。将蛋白质样品装载到凝胶上。</li>
		<li>通过将 DC 电源设置为10分钟, 然后 150 v 为45分钟, 运行 SDS 页面, 直到样品在凝胶底部的约1厘米处运行为止。</li>
		<li>染色 SDS 页凝胶与考马斯蓝色 (0.25% (瓦特/v) 考马斯明亮的蓝色 R250, 30% (v/v) 甲醇, 10% (v/v) 乙酸), 1 h。</li>
		<li>染色程序后, 去除考马斯蓝染色, 并应用去除染色缓冲器 (30% (v/v) 甲醇, 10% (v/v) 醋酸) 到蛋白质凝胶 > 12 小时室温。</li>
		<li>在白光下记录下凝胶以确认在 52 kDa 带 HEVNP ORF2 的存在。</li>
	</ol>
</li>
	<li>用 TEM 观察 HEVNPs。<ol>
<li>将 HEVNP 样品准备或稀释为 0.5-2 毫克/毫升, 10 毫米 MES pH 6.2 用于 TEM 成像。</li>
		<li>电离 40 mA 辉光放电的碳涂层网格在三十年代生产一个亲水性的碳表面。辉光放电设备在材料表中描述。 注: 在辉光放电处理后, 网格的亲水性碳表面只能持续30分钟。</li>
		<li>按住镊子, 在网格中添加2µL 的 HEVNP 样品, 等待15三十年代, 并用滤纸涂抹。</li>
		<li>立即用 ddH20 清洗网格, 然后用过滤纸涂抹。</li>
		<li>立即在网格中加入2µL 2% 的醋酸铀, 等待十五年代, 然后用过滤纸涂抹。将样品栅格放在电子除湿干燥柜中, 使其干燥。</li>
		<li>将网格转换成透射电镜和10-80k 放大图像。HEVNPs 出现在透射电镜作为空的 icosahedral 蛋白, 是27毫微米直径, 由于缺乏病毒 RNA。</li>
	</ol>
</li>
</ol>4. HEVNPs 与生物素、肿瘤靶向配体、显影的化学共轭
<ol><li>执行一步共轭 HEVNPs 和 maleimide 链接生物素。<ol>
<li>缓冲变化: 根据制造商的协议 (材料表), 将 HEVNPs 在迷你透析单元和透析中应用于室温下的0.01 米 PBS pH 值为1小时。将 HEVNPs 转移到1.5 毫升管, 用分光光度计测量 280 nm 的蛋白质浓度。</li>
		<li>将 HEVNP 混合到1毫克/毫升, 相当于18.8 µM 的胱氨酸反应站点 (参见步骤2.2.4 中的详细信息), 在0.01 米 PBS, pH 为 7.4, 以相等的 maleimide-生物素 (100 µM), 使1:5 摩尔比;反应4摄氏度。根据制造商的协议 (材料表), 删除未绑定 maleimide-生物素与 40 K 截留分子量自旋脱盐塔工艺。</li>
		<li>通过标准的减少 SDS 页面 (步骤 3.1) 分析样品。</li>
		<li>使用标准程序, 用酶联的链亲和素制备一种化学发光印迹。通过 x 射线胶片捕获化学发光信号 (图 2)。</li>
	</ol>
</li>
	<li>在混合动力核动力源上执行两步 LXY30 共轭到表面暴露的半胱氨酸 (图 5)。<ol>
<li>缓冲交换: 应用 HEVNPs 在微型透析单位和透析对 0.01 M PBS pH 值7.4 在室温下为 1 h. 将 HEVNPs 转移到1.5 毫升管, 用分光光度计测定 280 nm 的蛋白质浓度。</li>
		<li>添加650µM maleimide-叠氮化物和650µM alkyne-LXY3010在 0.01 M PBS pH 7.4 与200µM 丘索4和1毫米抗坏血酸形成 maleimide 链接的 LXY30 (Mal-LXY30) 在650µM. 孵育混合物在4°c 为 O/N。</li>
		<li>混合 HEVNP 到1毫克/毫升, 相当于18.8 µM 的胱氨酸反应站点 (见细节在步骤 2.2.4), 约10% 卷 Mal-LXY30 (650 µM) 在0.01 米 PBS pH 7.4, 使一个1:3 摩尔比率;反应4摄氏度。 注: 由于 Mal-LXY30 浓度相对较高, 反应物的最终浓度 (如丘索4) 在混合后减少约10倍, 以避免其对 HEVNPs 的损害。另一个选项是无铜共轭方法15。</li>
		<li>根据制造商的协议删除未绑定 maleimide-click-LXY30 与 40 K 截留分子量自旋脱盐塔 (请参阅材料表)。保持 LXY30-linked HEVNPs (LXY30-HEVNPs) 在4摄氏度。</li>
	</ol>
</li>
	<li>执行一步共轭 LXY30-HEVNPs 和 Cy5.5 nhs 酯 (nhs-Cy5.5)<ol>
<li>混合 LXY30-linked HEVNPs (LXY30-HEVNPs) 到1毫克/毫升, 这相当于胱氨酸反应站点的18.8 µM (参见步骤2.2.4 中的详细信息), 在0.01 米 PBS (100 Cy5.5) 的同等数量的 NHS-µM 的 pH 7.4, 使1:5 摩尔比率;反应4摄氏度。</li>
		<li>根据制造商的协议, 删除未绑定 Cy5.5-NHS 与40K 截留分子量自旋脱盐塔程序 (请参阅材料表)。保持 LXY30, Cy5.5 链接 HEVNPs (LXY30 HEVNP Cy5.5) 在4摄氏度。</li>
	</ol>
</li>
</ol>5. HEVNP 对 MDA-MB231 乳腺癌细胞的束缚和内化
<ol><li>种子 MDA-MB231 乳腺癌细胞成一个35毫米的玻璃底部菜肴 (5 x 104每碟) O/N 在哺乳动物细胞培养柜。</li>
	<li>对于细胞结合实验, 请按照步骤 4.2-4.3 准备 LXY30-HEVNP Cy5.5。稀释 LXY30-HEVNP-Cy5.5 0.01 毫克/毫升, 相当于 HEVNP ORF2 的0.188 µM (指步骤 2.2.4), 250 µL 0-1% 的血清/DMEM。<ol>
<li>在0.01 毫克/毫升 HEVNP Cy5.5 的情况下, 按步骤 4.3, 但与 NHS-Cy5.5 染料共轭, (HEV-Cy5.5), 准备阴性对照样品。稀释 HEVNP-Cy5.5 到0.01 毫克/毫升, 相当于 HEVNP ORF2 的0.188 µM (指步骤 2.2.4), 250 µL 10% 的血清补充 DMEM。</li>
	</ol>
</li>
	<li>通过应用250µL 1 米 PBS 缓冲液, pH 值7.4 来清洗细胞。清洗后取出 PBS 缓冲器, 同时在细胞培养皿中保留一些缓冲液。</li>
	<li>应用250µL 的 LXY30-HEVNP Cy5.5 在10% 的血清补充 DMEM 或 HEVNP Cy5.5 在10% 的血清补充 DMEM 的培养 MDA-MB231 乳腺癌细胞。用铝箔保护细胞培养皿。</li>
	<li>将细胞培养皿保持在37摄氏度细胞培养柜内, 用于内部化1小时。</li>
	<li>在冰上冲洗培养的细胞3次, 每洗5分钟, 250 µL 1 米 PBS, pH 7.4。</li>
	<li>在1米 pbs 中固定4% 只粉煤灰中的细胞, ph 7.4 为20分钟, 然后用250µL 的1米 pbs, ph 7.4 清洗一次。 注意: 细胞现在已经准备好被共聚焦显微镜成像。代表性数据显示在图 5中。</li>
</ol>

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作者声明他们没有竞争的兴趣。

与耗时的基因工程程序, 这通常需要几个星期, 在这里我们演示简单的两步和一步化学共轭程序, 可以在3天内完成, 增加癌症靶向配体和/或荧光检测染料到胱氨酸/赖氨酸 HEVNPs 的部位。该技术可用于筛选候选池中的最佳配体目标, 从而在合理的成本和交付时间内利用可用的肽/小分子合成服务。
不同于传统的基因工程, 它只能将多肽插入到感兴趣的蛋白质中, 化学共轭可以将各种...

在治疗和诊断分娩中纳米载体的发展, 被称为 nanotheranostics, 已经把大部分生物医学领域从广义治疗转移到目标传递1。靶向 nanotheranostic 交付将纳米载体 (纳米粒子) 与 theranostic 分子结合在一起, 稳定地直接 theranostic 分子到特定患病组织或生物化学途径2,3,4.纳米医学已经走到了目标交付的前列, 因为最佳尺寸的纳米粒子有能力稳定 theranostic 分子的循环, 有选择地靶向细胞表面分子呈现在患病的组织上。许多 nanotheranostic 平台仍然遭受被动细胞吸收, 前成熟退化, 毒性和与 theranostic 分子不充分的联系。VLPs 克服了目标交付中的许多障碍。它们被用作 nanocarriers 来显示外来的表位和/或提供小分子: 一种可以用来对抗许多疾病的方案1。该应用主要依赖于自组装的特性以及遗传修饰的方便性, 以满足给定 vip 的设计应用。与基因工程相比, 国外多肽对 vip 的化学共轭显示了一个重要的优势, 因为它允许多种实体, 如多肽或寡糖, 被共轭到 VLPs 的表面, 在调制和灵活的方式, 不改变贵宾大会。
HEVNPs, 来自重组的混合动力衣壳蛋白, 2nd开放阅读框架 (ORF2), 是非传染性的, 自组装衣壳能够细胞结合和进入。由于混合动力汽车进化为黏膜传输, 组装的衣壳蛋白是同样稳定的水解和酸性粘膜条件5。HEVNPs 形成一个空心, T = 1 icosahedral 衣壳, 由60个相同的单位组成,6,7 ORF2, 使其在储存和严酷的生理条件下高度稳定。由于缺乏任何病毒遗传元素, 通过昆虫细胞的杆状病毒表达系统实现了高效、高产的生产。由于其蛋白水解稳定性, 自组装 HEVNPs 提取和纯化的细胞上清, 大大减少必要的净化步骤。此外, HEVNPs 拥有一个表面暴露的凸出域 (P 域) 通过一个灵活的铰链连接到一个稳定的 icosahedral 基础。p 域在 icosahedral 基础上形成表面暴露的尖峰, 而柔性铰链使得在不损害基 icosahedral 结构的情况下可以显著地修改 P 域。与60个重复的单位, 单一站点特定的修改结果在60个对称站点的化学调制。最近, 我们提出了一个纳米平台使用 HEVNP, 可以化学共轭配体或小分子的 theranostic 应用。这是通过取代单一氨基酸与半胱氨酸的突出领域的混合动力-vip 作为一个反应地点与 maleimide 链接的肽或分子。基于以前对混合动力-vip 和研究良好的免疫表位8,9的结构分析, 以下五个 hev-vip 氨基酸以半胱氨酸取代为潜在候选者: Y485C、T489C、S533C、N573C 和 T586C (图 1)。通过透射电镜 (TEM) 观察 (图 2) 对昆虫细胞的表达和纯化后的 vip 形成进行了验证, 并在 maleimide 链接生物素后对暴露的半胱氨酸部位进行了分析。共轭 (图 2)。在五变种人中, HEVNP-573C 显示了 maleimide-生物素共轭 (图 2) 的最强信号, 并被用来作为乳腺癌细胞靶向4 (图 3) nanocarrier 的后续演示。
该协议描述了化学共轭方法, 通过表面半胱氨酸共轭将肿瘤靶向分子附着在 HEVNPs 上。我们详细的肿瘤靶向和检测分子结合的肿瘤传递与重组 HEVNPs 含有半胱氨酸在 N573 (HEVNP-573C)。我们专注于两步点击化学共轭过程, 以绑定乳腺癌肿瘤靶向肽, LXY3010到 HEVNPs 形成 LXY30-HEVNP (图 4)。随后, n-hydroxysuccimide (NHS)-Cy5.5 被共轭到单独的赖氨酸站点在 HEVNPs 建立 LXY30 HEVNP-Cy5.5 为荧光检测的在体外(图 5) 和在体内4.

<table><tbody><tr><td>MINI Dialysis Units, 10K MWCO</td><td>Thermo Fisher Scientific</td><td>69572</td><td>mini dialysis unit</td></tr><tr><td>High Five Cells</td><td>Thermo Fisher Scientific</td><td>B85502</td><td>Tn5 cells</td></tr><tr><td>SF9 Cells&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11496015</td><td>Sf9 cells</td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Thermo Fisher Scientific</td><td>A11101, A11100</td><td>Baculovirus expression system</td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Life Technologies</td><td>10359-016, 10360-014, 10584-027, 10712-024</td><td>Bacmid</td></tr><tr><td>ESF921 Insect Cell Media</td><td>Expression Systems LLC</td><td>96-001-01</td><td>insect cell media</td></tr><tr><td>Cy5.5 NHS ester, 5mg</td><td>Lumiprobe Corp</td><td>27020</td><td>Cy5.5 NHS ester</td></tr><tr><td>Zeba Spin Desalting Columns, 40K MWCO, 0.5 mL</td><td>Thermo Scientific</td><td>87766</td><td>spin desalting column</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich Chemical Co</td><td>M8250-250G</td><td>MES</td></tr><tr><td>Ultra-Clear Centrifuge Thinwall Ultra-Centrifuge Tubes</td><td>Beckman Coulter, Inc</td><td>Depends on Rotor</td><td>ultracentrifuge tube</td></tr><tr><td>NuPage 4-12% Bis-Tris Protein Gels</td><td>Thermo Fisher Scientific</td><td>NPO321BOX</td><td>SDS protein gel</td></tr><tr><td>Cellfectin II Reagent</td><td>Thermo Fisher Scientific</td><td>10362100</td><td>transfection reagent</td></tr><tr><td>EMS Glow Discharger</td><td>Electron Microscopy Science</td><td></td><td>glow discharger</td></tr></tbody></table>

surface functionalization of hepatitis e virus nanoparticles using chemical conjugation methods

病毒样粒子 (VLPs) 被用作 nanocarriers, 用于显示异物抗原和/或在各种疾病的检测和治疗中提供小分子。该应用依赖于基因修饰、自组装和半胱氨酸共轭来实现重组 VLPs 的肿瘤靶向应用. 与单基因修饰相比, 国外多肽对 VLPs 的化学共轭提供了一种很大的优势, 因为它允许多种实体, 如合成肽或寡糖, 以调制和灵活的方式共轭到 VLPs 表面, 而不改变 vip 组装。
在这里, 我们演示如何使用 E 型肝炎病毒纳米粒子 (HEVNP), 一个模块化的 theranostic 胶囊, 作为一个多功能运送载体。HEVNPs 的功能包括组织靶向、影像学和治疗性分娩。在 HEVNP 的结构研究基础上, 选取了结构上独立的和表面暴露的残留物作为 maleimide 连接化学基团的共轭点, 通过硫醇选择性的联系来选择半胱氨酸置换。一种特殊的半胱氨酸修饰 HEVNP (胱氨酸取代天门冬在 573 aa (HEVNP-573C)) 被共轭到乳腺癌细胞特异配体, LXY30 和标记为近红外线 (近红外) 荧光染料 (Cy5.5), 呈现肿瘤靶向HEVNPs 作为有效的诊断胶囊 (LXY30-HEVNP Cy5.5)。类似的工程策略可以与其他大分子复合物与众所周知的原子结构, 以探索潜在的应用 theranostic 交付。

类似于 HEV-VLPs, 所有胱氨酸改性 HEVNPs 形成可溶性 icosahedral 衣壳, 并没有聚集在溶液中的生产或纯化。在单步 maleimide-生物素共轭之前和之后, 每个胱氨酸修改后的 HEVNPs 在负染色 EM (图 2) 中都与混合动力 VLPs 不区分。Maleimide-生物素共轭效率胱氨酸改性 HEVNPs 首次测试与西方印迹通过化学发光链亲和素结合。继 maleimide-生物素共轭后, 胱氨酸修饰 HEVNPs 显...

Watch this Scientific Journal Video about Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods at JoVE.com

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

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

我们已将戊型肝炎病毒的衣壳蛋白作为 theranostic 纳米微粒 (HEVNP) 进行了设计。HEVNP 自组装成一个稳定的 icosahedral 笼在黏膜分娩。在这里, 我们描述了 HEVNPs 的改变的肿瘤靶向性的变异表面暴露残留到半胱氨酸, 共轭合成配体, 具体绑定肿瘤细胞。

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

surface-functionalization-hepatitis-e-virus-nanoparticles-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.8K 次观看.  University of California Davis. 我们已将戊型肝炎病毒的衣壳蛋白作为 theranostic 纳米微粒 (HEVNP) 进行了设计。HEVNP 自组装成一个稳定的 icosahedral 笼在黏膜分娩。在这里, 我们描述了 HEVNPs 的改变的肿瘤靶向性的变异表面暴露残留到半胱氨酸, 共轭合成配体, 具体绑定肿瘤细胞。

视频: Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

科研

免疫与感染

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 ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

癌症研究

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

功能化磁性纳米粒子的合成及其与侧双磷铁氧胺的结合及其细菌检测评价

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

化学

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

一种通过牛磺胆酸钠共转运多肽作为治疗靶点检查乙型肝炎病毒进入的感受态肝细胞模型

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

生物化学

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

生物工程

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

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

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they&#39;re highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Q&#946;. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

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

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

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

磁性纳米材料的生物功能化

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

通过表面等离子体共振 设计 抗病毒药物

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

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

摘要

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此视频中的章节

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

通过Bioorthogonal点击化学化学选择性病毒表面改性

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

功能化磁性纳米粒子的合成及其与侧双磷铁氧胺的结合及其细菌检测评价

一种通过牛磺胆酸钠共转运多肽作为治疗靶点检查乙型肝炎病毒进入的感受态肝细胞模型

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

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

磁性纳米材料的生物功能化

通过表面等离子体共振设计抗病毒药物

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

摘要

探索更多视频

此视频中的章节

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

通过Bioorthogonal点击化学化学选择性病毒表面改性

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

功能化磁性纳米粒子的合成及其与侧双磷铁氧胺的结合及其细菌检测评价

一种通过牛磺胆酸钠共转运多肽作为治疗靶点检查乙型肝炎病毒进入的感受态肝细胞模型

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

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

磁性纳米材料的生物功能化

通过表面等离子体共振 设计 抗病毒药物

通过表面等离子体共振设计抗病毒药物