Name: rapid, seamless generation of recombinant poxviruses using host range and visual selection
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1. 生成重组矢量
<ol><li>设计引漆以生成选择盒。使用多个在线引物设计工具，使用多个在线引物设计工具中的任何一种具有重叠序列和矢量设计每个具有重叠序列的单个放大器，以方便 DNA 分子的等温酶组装，也称为 Gibson 组装。 注：此协议也可以使用传统的基于限制内核酶的克隆方法完成。在这种情况下，使用适当的限制位点而不是重叠序列设计引素。</li>
	<li>使用步骤 1.1 中设计的引素，PC...

<ol>
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在这里，我们提出了一个瞬态标记选择策略32的变化，以生成重组性气喘病毒，而不保留外来DNA在最后重组病毒。我们的策略使用由宿主抗病毒蛋白PKR介导的选择性压力，而不是其他形式的选择性压力，如抗生素。使用宿主抗病毒基因消除了细胞中化学诱导的型物变化的可能性，或由于选择药物而增加突变的风险。此外，与药物选择不同，我们的方法没有滞后阶段，因为PKR在所?...

Vaccinia病毒（VACV）有助于首次成功地从自然界中消灭人类病原体变异病毒（VARV）。自从病毒灭绝以来，包括VACV在内的痘病毒一直为人类和动物医学提供有用的治疗病毒。例如，基于VACV的狂犬病病毒疫苗在防止神经病性狂犬病在欧洲1和美国2的传播方面非常有效。最近，反应各种抗肿瘤分子（如单链抗体或人类红细胞生成素）的重组痘病毒作为溶血剂33、4、54,5取得了令人鼓舞的成功。VACV作为载体特别有吸引力，因为它容易受遗传操作，具有广泛的宿主范围，在各种条件下保持稳定，便于运输和疫苗在6、77领域的生存能力。虽然已经开发出多种技术来生成用于实验室实验和疫苗生成的重组VACV，但目前产生这些病毒的策略有显著的局限性。
由于VACV的效用，开发了多种生成重组病毒的策略。第一种策略采用同源重组来引入一种盒式磁带，包括转基因和可选标记基因，如抗生素耐药性基因。盒式磁带的两个+500核苷酸（nt）或更大的手臂将基因定向到病毒基因组中的特定部位，然后通过双交叉事件88、9、109,10进行稳稳地集成。这一战略是快速和高效的;然而，它产生额外的遗传物质的形式标记基因，可能产生意想不到的效果。此外，转基因数量有实际的上限，可以引入的转基因标记数量受可用独特可选择标记的数量限制。瞬时主导选择（TDS）策略通过促进"无痕"重组病毒的生成解决了这一问题。使用这种策略，包含突变VACV基因和可选择标记基因的质粒被集成到病毒基因组中，但没有额外的侧翼VACVDNA。这种方法导致整个质粒的暂时整合和VACV基因的重复，由于单个交叉事件的整合。这种中间体是稳定的，只要它在选择压力下保持，允许此构造的丰富。移除选择后，VACV 复制启用第二个交叉事件，导致以大约 50：50 的比例去除质粒并随后形成野生类型 （wt） 或重组病毒。虽然TDS生成重组病毒而无需稳定引入外来DNA，但多个病毒克隆必须通过测序分析筛选出预期的突变，这一步骤可能耗时且成本高昂。
在这里，我们提出了一种生成重组痘病毒的方法，结合每种方法的最佳方面，类似于为复制无能的、经过修改的安卡拉12、13、14,14所述的方法。12,此策略结合了视觉和宿主范围选择，通过双交叉事件快速生成重组病毒，然后通过同源重组消除可选标记基因。这种方法允许由同源重组促进的突变体的快速生成，具有TDS方法的"无痕"性质，同时无需随后的筛选步骤来区分野生病毒和突变病毒。我们的方法还采用宿主范围选择代替抗生素选择，消除了细胞系中化学诱导的型比变化的风险。对于这种方法，我们选择使用宿主抗病毒蛋白激酶R（PKR）作为选择性剂，以产生重组VACV。在大多数单元格类型15中，PKR 表示为非活动单体。在N-终端dsRNA结合域中结合双链RNA（dsRNA）时，PKR变暗，并自磷化16。这种活性形式的PKR磷酸酶酶启动因子2（eIF2）的α亚单位，最终抑制启动器二甲酰-tRNA的传递，从而防止细胞内翻译，并广泛抑制许多病毒家族17，18,18的复制。
为了应对PKR广泛而有效的抗病毒活性，许多病毒至少进化了一种防止PKR激活的策略。大多数痘病毒表达两个PKR拮抗剂，由VACV中的E3L和K3L基因编码，通过两种不同的机制19对抗PKR。E3通过结合双链RNA20，2121来防止20PKR消增，而K3则通过直接结合到激活的PKR，从而抑制与其基质eIF2+22的相互作用，作为伪22基质抑制剂。重要的是，这两个PKR拮抗剂不一定能抑制所有物种的PKR。例如，羊痘病毒的K3同源性强抑制羊的PKR，而羊痘E3同源性没有表现出相当大的PKR抑制23，24。23,在这项研究中，我们提出了一种使用PKR介导选择性压力与荧光选择相结合的方法，为E3L和K3L（VC-R4）生成一种VACV重组，这种重组不能在从不同物种衍生的PKR合格细胞中复制。这种重组病毒为快速生成重组病毒提供了极好的背景，这些病毒表达由原生E3L启动器控制的基因。

<table>
	<tbody>
		<tr>
			<td>2X-Q5 Master Mix</td>
			<td>NEB</td>
			<td>M0492L</td>
			<td>High-fidelity polymerase used in PCR</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>ThermoFisher Scientific</td>
			<td>11593027</td>
			<td>Bacterial selective agent</td>
		</tr>
		<tr>
			<td>Disposable Cell Scrapers</td>
			<td>ThermoFisher Scientific</td>
			<td>08-100-242</td>
			<td>Cell scraper to harvest infected cells</td>
		</tr>
		<tr>
			<td>EVOS FL Auto 2 Cell imaging system</td>
			<td>ThermoFisher Scientific</td>
			<td>AMAFD2000</td>
			<td>Fluorescent microscope</td>
		</tr>
		<tr>
			<td>EVOS Light Cube, GFP</td>
			<td>ThermoFisher</td>
			<td>AMEP4651</td>
			<td>GFP Cube</td>
		</tr>
		<tr>
			<td>EVOS Light Cube, RFP</td>
			<td>ThermoFisher</td>
			<td>AMEP4652</td>
			<td>RFP Cube</td>
		</tr>
		<tr>
			<td>GenJet</td>
			<td>SignaGen Laboratories</td>
			<td>SL100489</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Luria Bertani (LB) Broth</td>
			<td>Gibco</td>
			<td>10855021</td>
			<td>Bacterial growth medium</td>
		</tr>
		<tr>
			<td>Monarch DNA gel extraction kit</td>
			<td>NEB</td>
			<td>T1020L</td>
			<td>Gel purification kit used to purify amplicons and linearized vectors</td>
		</tr>
		<tr>
			<td>Monarch Plasmid Miniprep kit</td>
			<td>NEB</td>
			<td>T1010L</td>
			<td>Miniprep kit ussed to purify plasmids</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-ONE-W</td>
			<td>Spectrophotometer used to measure RNA and DNA concentration</td>
		</tr>
		<tr>
			<td>NEBuilder Master Mix</td>
			<td>NEB</td>
			<td>E2621L</td>
			<td>Isothermal enzymatic assembly kit used to generate the recombination vector</td>
		</tr>
		<tr>
			<td>Q500 Sonicator</td>
			<td>Qsonica</td>
			<td>Q500-110</td>
			<td>Sonicator for virus lysates</td>
		</tr>
		<tr>
			<td>RK13 cells</td>
			<td>ATCC</td>
			<td>CCL-37</td>
			<td>Rabbit kidney cells</td>
		</tr>
		<tr>
			<td>VWR Multiwell Cell Culture plates</td>
			<td>VWR</td>
			<td>10062-892</td>
			<td>Cell culture plates</td>
		</tr>
	</tbody>
</table>

rapid, seamless generation of recombinant poxviruses using host range and visual selection

Vaccinia病毒（VACV）有助于从自然中根除天花的病原体变异病毒（VARV）。VACV自首次作为疫苗使用以来，一直作为治疗性疫苗的载体和溶血性病毒而开发。这些应用利用 VACV 易于操作的基因组和广泛的主机范围，作为一个出色的平台，以生成具有各种治疗应用的重组病毒。已经开发了几种方法来生成重组VACV，包括标记选择方法和瞬态显性选择。在这里，我们介绍了主机范围选择方法的改进，以及重组病毒的可视化识别。我们的方法利用宿主抗病毒蛋白激酶R（PKR）产生的选择性压力，加上荧光融合基因，表达mCherry标记的E3L，这是两个VACV PKR拮抗剂之一。盒式磁带，包括感兴趣的基因和mCherry-E3L融合，由从VACV基因组派生的序列组成。感兴趣的基因和mCherry-E3L是一个较小的区域，与3'臂的前150个核苷酸相同，促进mCherry-E3L基因在选择后的同源重组和丢失。我们证明，这种方法允许在各种细胞类型中高效、无缝地生成 rVACV，而无需选择药物或广泛筛选突变病毒。

我们使用图 1中所示的过程生成了一个 VACV，该 VACV 缺少 PKR 拮抗器 E3L 和 K3L，在已为 K3L （vP872） 删除的病毒中用 EGFP 替换 E3L。图 2显示了 PKR 合格 RK13 细胞中指示 mCherry-E3L 病毒表达的红色荧光斑块，以及 RK13_E3L_K3L 细胞中的 EGFP，确认 E3L 的丢失和 mCherry-E3L 选择标记的崩溃。图3证实，这种重组病毒VC-R4，缺少两个PKR拮抗剂不能复?...

Watch this Scientific Journal Video about Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection at JoVE.com

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

这是一种使用宿主范围选择和重组病毒的可视化识别来生成"无疤痕"重组病毒的方法。

使用主机范围和视觉选择快速、无缝地生成重组痘病毒

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a ...

我们的方法使用主机范围选择和荧光标记，在各种细胞类型中高效无缝地生成重组病毒。该技术将同源重组的速度与瞬态显性选择的无疤痕性质相结合。此外，我们托管范围选择，以消除化学诱发变化的可能性。这种方法还可用于修改瓦奇尼亚病毒或其他痘病毒，以表达异种基因，例如，生成疫苗。如果您仅使用荧光选择执行此协议，则必须确保筛选的病毒足以检测相对罕见的不 PKR 选择性压力的重组剂。此方法依赖于荧光标记的增益和丢失，以成功选择已获得重组盒的病毒，并检测具有无疤痕重组的病毒。为了生成重组载体，首先将17微升DNase无水、每个底面的1.2微升、5x PCR反应缓冲液的5微升、模板DNA和0.5微升DNA聚合酶添加到每安培的PCR管中。添加额外的无德纳塞水，使每个管的最终体积达到50微升。将管子放入热循环器中。在98摄氏度下熔化DNA30秒，然后使用25轮三步PCR协议。要可视化 1%agarose 凝胶上的扩增产品，请向每个井中加入 10 微升每个 DNA 产品和 2 微升的加载缓冲液。以每厘米八伏运行凝胶一小时。在运行结束时，使用脱氧核糖核酸凝胶提取套件，根据制造商的协议凝胶净化每个安培。用 50 微升的 DNase 无水和立即离心从柱上挖出安普利森。要使克隆载体线性化，首先在37摄氏度下将1微克的载体、17微升的DNase无水、2微升的反应缓冲液和1微升的EcoRI添加到管中，进行一小时的孵育。在孵育结束时，将线性化载体10微升的DNase自由水和10微升的DNA组装主混合物添加到每个分离的安培体中。在孵化结束时，添加0.2个picomoles。在50摄氏度下孵育样品一小时。在孵化结束时，根据标准协议，用两个微升的组装产品将化学合格的大肠杆菌进行转化。板 100 微升转化细胞到 LB agarose 板上， 每毫升安他西林补充 100 微克。在37摄氏度下过夜孵育后，选择分离良好的菌落在Luria肉汤中接种，并在37摄氏度和每分钟225转的一夜之间每毫升安霉素100微克补充。第二天早上，使用质粒微型组件套件来隔离质粒。并在分光光度计上检查DNA的浓度和纯度。对于重组病毒生成，感染一个合适的细胞的汇合单层与病毒重新组合在一个六井板中的一个感染的多重。在37摄氏度和5%的二氧化碳下孵育受感染的细胞一小时。在孵化结束时，用新鲜的DMEM更换每一个井中的上经剂，并按照制造商的协议使用市售的转染试剂。向每一井添加三微克的重组向量。将板放在培养箱中，额外放置 48 小时。在将每个转染条件的细胞汇集到单独的 1.5 毫升管中之前。然后冷冻三次池细胞，然后以50%的振幅对所得的乳酸盐进行15秒的声波化。接下来，连续10倍稀释声波化的利沙酸盐，从10至第一至10至第六浓度新鲜DMEM，并添加1毫升每稀释到蛋白质激酶R能力细胞系的个别汇合井。将细胞放在细胞培养箱中一小时，然后用新鲜培养基取代上位，将细胞返回到细胞培养箱。24 至 48 小时后，通过荧光显微镜识别重组病毒。由于mCherry-E3L融合基因的整合，重组病毒的斑块表达红荧光。斑块在野生型RK13细胞上三次净化重组病毒。经过三轮斑块纯化后，用斑块的序列稀释菌斑菌感染RK13+E3L+K3L细胞。要识别折叠的病毒，使用配备GFP和RFP滤波块的适当成像系统进行荧光显微镜。然后斑块纯绿色VC-R4，或无色E3L斑块在RK13+E3L+K3L细胞上三次。确保没有斑块发红。感染细胞与至少100个斑块形成单位，以增加你识别无色斑块的机会。在极少数情况下，可能需要多轮感染。蛋白质激酶R RK13细胞中的红色荧光斑块是mCherry-E3L病毒表达的指标。在RK13+E3L+K3L细胞中只表达eGFP的无色斑块或斑块证实了mCherry-E3L选择标记的崩溃。重组病毒VC-R4，缺乏两种蛋白质激酶R拮抗剂不能在蛋白激酶R能力RK13细胞中复制，而表观的病毒VP872，表示E3L是复制能力。为了确认这种无法在RK13细胞中复制，仅是由于E3L的丢失，增强型GP被VC-R4病毒中的E3L所取代。它使用相同的选择协议生成相关病毒。有趣的是，在制造这种病毒时，在选择RK13+E3L+K3L细胞之前，在选择通常需要选择无疤痕重组剂时，会识别到与mCherry-E3L选择标记折叠一致的无色斑块。可能是因为 mCherry-E3L 重组盒和插入 VC-R4 的 E3L 基因之间的扩展序列标识。平均而言，12.6%的后代病毒与转染质粒进行了重组，与先前报告的频率相似。在这里，无色斑块相对于总斑块的频率RK13+E3L+K3L细胞，表明塌陷率和mCherry-E3L选择标记的丢失发生在大约1.8%的频率。在第一阶段使用PKR功能细胞确保所有含有重组病毒的斑块。在第二阶段使用PKR缺陷细胞，可以检测重组病毒与崩溃mCherry-E3L位点。这种方法使我们能够快速生成含有外源基因的病毒，例如，疫苗生产提供不同病毒PKR拮抗剂的表达。促进分析不同宿主与PKR的物种特定相互作用。

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

链霉菌发育突变体的视觉和显微评价

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

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, ...