Name: growth, purification, and titration of oncolytic herpes simplex virus
Uploaded: 2021-05-13T14:30:00
Description: Watch this Scientific Journal Video about Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus at JoVE.com

萨哈实验室的研究部分得到了美国劳工部（W81XWH-20-1-0702）和道奇琼斯基金会-阿比林的资金支持。塞缪尔·拉布金和梅丽莎·汉弗莱.M部分得到国家卫生研究院（R01 CA160762）的支持。

1） 奥赫斯夫增长
注：在与 oHSV 合作之前，确保机构生物安全委员会的批准。这项研究是根据经核准的IBC第18007号议定书进行的。保持BSL2预防措施：漂白所有与病毒接触的管道、尖端、管子和其他材料。在手离开BSL2细胞培养罩之前，用70%异丙醇喷洒手套。使用病毒后，始终用肥皂水彻底洗手。
<ol><li>在第 -1 天，种子低通道 Vero 细胞在 20 T-150 厘米2 烧瓶密度为 7-8 × 1...

<ol>
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该协议从低通道维罗细胞中的 oHSV 生长开始。在病毒接种时，Vero细胞单层的对流应为+80%，因为过度生长的细胞可以发展出紧密的纤维结构，减少OHSV进入Vero细胞20。观察到 90-100% CPE 后，培养超自然体被去除，细胞被收获，在 VB/超自然剂中再悬浮（参见步骤 1.4.6），快速冷冻，并储存在 -80 °C 以供以后净化。Blaho和他的同事采用了一种略有不同的方法来采集和储存受感染的维罗细...

肿瘤病毒（OVs）是癌症免疫治疗的一种新兴和独特的形式。OVs有选择地复制和解毒肿瘤细胞（备用正常/健康细胞）1，同时诱导抗肿瘤免疫2。单纯疱疹病毒（oHSV）是所有OV中研究最广泛的病毒之一。这是最远的沿诊所， 与塔利莫金拉赫帕雷普韦克 （T-VEC） 是第一个也是唯一的 OV 获得 FDA 批准在美国治疗晚期黑色素瘤3.除了T-VEC，许多其他基因工程的oHSV正在临床和临床上测试不同的癌症类型3，4，5，6，7，8。目前先进的重组DNA生物技术进一步增加了工程新oHSV编码治疗转基因的可行性。在任何（新开发的）oHSV能够进行体外和体内研究测试之前，有效的 oHSV 传播、纯化和滴定测定系统至关重要。本文描述了一个简单的分步方法（在维罗细胞中）、纯化（通过蔗糖梯度法）和滴定（通过在维罗细胞中的 oHSV 斑块检测）（图 1）。它可以很容易地在任何生物安全级别 2 （BSL2） 实验室设置中采用，以实现高品质的病毒库存，用于前科研究。
维罗，非洲绿猴肾细胞系，是最常见的细胞系为oHSV传播9，10，11，12，13，因为维罗细胞有一个有缺陷的抗病毒干扰素信号通路14。其他具有干扰素基因（STING）信号的灭活刺激器的细胞系也可用于oHSV增长12，13。此协议利用 Vero 细胞进行 oHSV 生长和斑块检测。繁殖后，oHSV感染的细胞被收获、解毒并接受净化，其中被浸渍细胞首先用苯甲酶核酶处理，以降解宿主细胞DNA，防止核酸蛋白聚集，降低细胞的粘度。由于适当激活苯甲酶通常需要毫克2+，1-2 mm MgCl2用于此协议15。在高速蔗糖梯度离心之前，通过连续过滤进一步消除苯甲酸酯处理细胞裂解物中的宿主细胞碎片。粘性25%蔗糖溶液垫有助于确保通过蔗糖层的病毒迁移速度更慢，将宿主细胞相关成分留在超自然体内，从而改善净化和限制颗粒16中的病毒损失。净化的 oHSV 然后滴定在 Vero 细胞上，病毒斑块通过 Giemsa 染色17或 X gal 染色（用于 LacZ 编码 oHSV）18来可视化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL centrifuge tubes</td>
			<td>Sigma</td>
			<td>CLS3620</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene tubes</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene centrifuge tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma</td>
			<td>E8263-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>Corning</td>
			<td>MT-10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>MT-21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH3007003</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa Stain</td>
			<td>Sigma</td>
			<td>G3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-262-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>MT-21-021-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Glucose Dulbecco&#8217;s Phosphate-buffered Saline</td>
			<td>Sigma</td>
			<td>D4031</td>
			<td></td>
		</tr>
		<tr>
			<td>Human immune globulin</td>
			<td>Gamastan</td>
			<td>NDC 13533-335-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Sterilization filter, 250 mL</td>
			<td>Nalgene, Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Sterilization filter, 500 mL</td>
			<td>Nalgene, Fisher Scientific</td>
			<td>09-740-25C</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red solution</td>
			<td>Sigma</td>
			<td>N4638</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher scientific</td>
			<td>&#160;15710S</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate rocker</td>
			<td>Fisher</td>
			<td>88861043</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferricyanide</td>
			<td>Sigma</td>
			<td>P8131</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferrocyanide</td>
			<td>Sigma</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75004381</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST 21R Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75002446</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes with Screw Caps</td>
			<td>Fisher Scientific</td>
			<td>02-681-371</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.45 PVDF</td>
			<td>MilliporeSigma</td>
			<td>SLHV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.8 MCE</td>
			<td>MilliporeSigma</td>
			<td>SLAA033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, 5 &#181;m PVDF</td>
			<td>MilliporeSigma</td>
			<td>SLSV025LS</td>
			<td></td>
		</tr>
		<tr>
			<td>T150 culture flask</td>
			<td>Falcon</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>MP Biomedicals LLC</td>
			<td>816116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic water bath</td>
			<td>Branson</td>
			<td>CPX-952-116R</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Corning</td>
			<td>46-101-RF</td>
			<td></td>
		</tr>
	</tbody>
</table>

growth, purification, and titration of oncolytic herpes simplex virus

肿瘤病毒（OVs），如肿瘤疱疹单纯病毒（oHSV），是癌症免疫治疗领域快速增长的治疗策略。OVs，包括oHSV，有选择地复制和杀死癌细胞（拯救健康/正常细胞），同时诱导抗肿瘤免疫。由于这些独特的特性，基于 oHSV 的治疗策略越来越多地用于癌症的治疗，临床前和临床上，包括 FDA 批准的塔利莫金·拉赫帕雷韦克 （T-Vec）。生长、纯化和滴定是任何 OEV（包括 oHSV）的三种基本实验室技术，在它们可用于实验研究之前。本文描述了一个简单的分步方法来放大维罗细胞中的 oHSV。当 oHSV 成倍增加时，它们在 Vero 细胞中产生细胞病变效应 （CPE）。一旦90-100%的受感染细胞显示CPE，它们就会被轻轻收获，用苯甲酸酯和氯化镁（MgCl2）进行处理，过滤后，使用蔗糖梯度法进行净化。净化后，传染性 oHSV（指定为斑块形成单位或 PPF）的数量由 Vero 细胞中的"斑块检测"确定。此处描述的协议可用于为细胞培养和体内动物实验的体外研究准备高滴度 oHSV 库存。

图 1中描述了整个协议的简要概述，该图代表了 oHSV 的生长、净化和认证的关键步骤。Vero细胞中的CPE最早可检测到4小时后HSV感染19。图2在 oHSV 感染后的三个不同时间点演示 Vero 细胞中的 CPE。随着时间的推移，CPE 的水平会提高。在此协议中，90-100% CPE 通常在低 MOI oHSV 接种的 48 小时内观察到（这是收获细胞进行净化的最佳时机）。但?...

Watch this Scientific Journal Video about Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus at JoVE.com

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

在本手稿中，我们描述了一个简单的方法的生长，纯化和滴定肿瘤疱疹简单病毒的药物前使用。

单纯疱疹病毒的生长、净化和滴定

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer ...

肿瘤性疱疹单纯病毒是癌症免疫治疗的一种新兴形式。有效的病毒传播、净化和三位一体测定系统对于在实验研究中使用至关重要。这种方法非常简单，可在任何生物安全二级实验室环境中轻松采用，以获得高质量的病毒库存进行前科研究。生产高质量、纯病毒性库存至关重要，因为它用于研究抗癌免疫反应，并开发一种基于病毒的新型癌症免疫疗法。此协议可用于净化任何野生类型或转基因疱疹简单病毒。对于从未执行过此技术的个人，此协议应易于阅读和遵循。上市材料和试剂应在第一次尝试此技术之前就位。首先用两倍高的葡萄糖 DPBS 清洗 T-150 平方厘米的烧瓶，并辅以 1% IFCS。吸气 DPBS，每瓶添加 7 毫升病毒接种。轻轻摇动烧瓶5分钟，在37摄氏度下孵育1.5至2小时。然后，取出接种，并在每个烧瓶中加入 25 毫升 DMEM，并辅以 1% IFCS。在37摄氏度和5%的二氧化碳孵化两到四天后，从每个烧瓶中收集20毫升培养超自然物，放入50毫升离心管中。使用细胞刮刀，从烧瓶底部刮出细胞。将先前收集的培养超标物约 15 毫升添加到每个烧瓶中，使体积达到 20 毫升，然后使用 10 毫升无菌血清制液处理器轻轻清洗烧瓶底部几次。将中等体收集到放置在冰上的50毫升圆锥形离心管中。在4摄氏度下，以300倍G分立管10分钟。将每个颗粒彻底补充到 1.25 毫升病毒缓冲器（VB）和 1.25 毫升以前收集的培养超自然物中。将所有重新喷出的颗粒混合到一个 50 毫升圆锥形离心机管中。使用干冰和100%乙醇将再喷出的细胞冷冻，并储存在零下80摄氏度。在37摄氏度的温水浴中解冻先前的冷冻细胞，在水浴中三次循环发出声波。加入175单位的本佐纳塞核糖和两微升的两毫升氯化镁每毫升细胞裂解和涡流。孵化30分钟后，在37摄氏度的温水浴中，将管子放在冰上。将细胞的利萨酸盐以300倍G旋转10分钟。在新的 50 毫升圆锥离心管中收集超细剂，并在 500 微升 VB 中重新注入细胞颗粒。指定它为弹丸一。以 500 倍 G 再次旋转收集的超纳特 10 分钟，并将超纳特分别存放在新鲜的 50 毫升圆锥离心管中，以便以后使用。将细胞颗粒重新喷入VB的500微升中，指定为2粒。将重新喷出的颗粒一和颗粒二混合在一个新的1.75毫升离心管和涡流中，在水浴中两次发出声波，然后以400倍G旋转管10分钟。从 1.7 毫升离心机管中收集超纳特，并与之前收集的 50 毫升圆锥离心管相结合。通过无菌的五微米PVDF膜过滤器将超自然体过滤成新的50毫米离心管，称为管一。将一毫升 VB 添加到 50 毫升离心机管中，在过滤前一步的超自然体涡流后排空，并通过放置在管一上的相同 PVDF 膜过滤器将其传递。将过滤液从管一通过无菌 0.8 微米 MCE 膜过滤器放置在称为管二的新型 50 毫米离心管上。将一毫升 VB 添加到空管一个涡流中，并通过放置在管二上的相同 MCE 过滤器将其传递。将过滤液从管二通过无菌 0.45 微米 PVDF 过滤器放置在新的 50 毫升离心机管标记管三。如前所述，重复用一毫升VB清洗管二，将过滤剂添加到管三。在这个具有代表性的分析中，在受感染细胞四舍五入后36小时、48小时和72小时，可以在Vero细胞中观察到细胞病变效应。随着时间的推移，CPE 水平的提高通过 mCherry 表达来明显可见。感染后72小时，病毒斑块被X-gal染色，用 lacZ 表达可视化 oHSV。轻轻擦洗病毒感染细胞，轻轻清洗烧瓶底部，对于保持所有HSV受感染细胞完好无损，从而获得高滴定病毒存量至关重要。

Research

JoVE Journal

Immunology and Infection

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Epstein - Barr病毒生长转化的淋巴细胞系的建立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Assembly and Purification of Prototype Foamy Virus Intasomes

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

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, ...