Name: surface functionalization of hepatitis e virus nanoparticles using chemical conjugation methods
Uploaded: 2018-05-11T14:00:00
Description: Watch this Scientific Journal Video about Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods at JoVE.com

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...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="984">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:391px;">MINI Dialysis Units, 10K MWCO</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:265px;">69572</td>
			<td style="width:167px;">mini dialysis unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Five Cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>B85502</td>
			<td>Tn5 cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SF9 Cells&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11496015</td>
			<td>Sf9 cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Bac-to-Bac Baculovirus Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11101, A11100</td>
			<td>Baculovirus expression system</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">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 height="20">
			<td height="20" style="height:20px;">ESF921 Insect Cell Media</td>
			<td>Expression Systems LLC</td>
			<td>96-001-01</td>
			<td>insect cell media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy5.5 NHS ester, 5mg</td>
			<td>Lumiprobe Corp</td>
			<td>27020</td>
			<td>Cy5.5 NHS ester</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeba Spin Desalting Columns, 40K MWCO, 0.5 mL</td>
			<td>Thermo Scientific</td>
			<td>87766</td>
			<td>spin desalting column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES Hydrate</td>
			<td>Sigma-Aldrich Chemical Co</td>
			<td>M8250-250G</td>
			<td>MES</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultra-Clear Centrifuge Thinwall Ultra-Centrifuge Tubes</td>
			<td>Beckman Coulter, Inc</td>
			<td>Depends on Rotor</td>
			<td>ultracentrifuge tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NuPage 4-12% Bis-Tris Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>NPO321BOX</td>
			<td>SDS protein gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellfectin II Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362100</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">EMS Glow Discharger</td>
			<td style="width:161px;">Electron Microscopy Science</td>
			<td style="width:265px;"></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。

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