这项工作得到了西华师范大学提供的博士生科研启动基金（编号 20E059）的财政支持。

1. PRRSV 基因模板的制备
<ol><li>在 1 mL RNA 提取试剂中解冻病毒原液（参见 材料表）。</li>
	<li>加入 0.2 mL 氯仿并充分混合。孵育 3 分钟。</li>
	<li>在 4 °C 下以 12,000 × g 离心混合物 15 分钟。 注:混合物分为三个相，即无色水相、间相和红色苯酚-氯仿相。</li>
	<li>吸出无色水相并将其转移到新管中。</li>
	<li>将水相与 0.5 mL 异丙醇充分混合，并在 4 °C 下孵育 10 分钟。</li>
	<li>在 4 °C 下以 12,000 × g 离心 10 分钟。 注:试管底部有白色 RNA 沉淀物。</li>
	<li>使用微量移液器丢弃试管的上清液。</li>
	<li>加入 1 mL 75% 乙醇以重悬沉淀并短暂涡旋。</li>
	<li>在 4 °C 下以 7,500 × g 离心 5 分钟。 使用微量移液器丢弃试管的上清液。</li>
	<li>将 RNA 干燥 5 分钟，加入 20-50 μL 不含 RNase 的水以重悬 RNA，并充分混合。</li>
	<li>继续进行逆转录;设置反应以进行逆转录，如 表 1 所示。 注:为确保逆转录成功，请使用高质量的 RNA 模板。</li>
	<li>使用所得的 cDNA 进行 PCR 或将其储存在 -20 °C。</li>
</ol>2. PCR 引物设计
<ol><li>设计正向引物<ol><li>打开软件并选择 New DNA File（新建 DNA 文件）。</li>
		<li>将 PRRSV 基因序列 （GenBank: FJ548852.1） 从 NCBI 粘贴到软件中。单击 OK 生成序列文件。</li>
		<li>分析序列并标记片段连接。设计片段的正向特异性序列引物。在大多数情况下，优选的熔解温度 （Tm） 在 55 °C 和 62 °C 之间，GC 含量为 40-60%。</li>
		<li>单击 Primers 并选择 Add Primer。</li>
		<li>粘贴特异性序列，并将载体的重叠序列添加到特异性序列引物的前 5' 核苷酸中。在每个连接附近，选择 20-40 bp 作为两个相邻片段之间的重叠区域。</li>
		<li>命名包含突出端和特定序列的正向引物（参见 补充图 S1）。</li>
		<li>单击 Add Primer to Template（将引物添加到模板）。</li>
	</ol></li>
	<li>设计反向引物<ol><li>在软件中分析序列并标记片段连接。设计片段的反向特异性序列引物。</li>
		<li>单击 Primers 并选择 Add Primer。</li>
		<li>粘贴特异性序列，并将限制性位点添加到特异性序列引物的前 5' 核苷酸上。 注:除多个克隆位点外，插入片段和载体中必须不存在添加的限制性位点。</li>
		<li>将 20-40 bp 的载体突出端序列添加到限制性位点的前 5' 核苷酸中。</li>
		<li>命名包含突出端和特定序列的反向引物（参见 补充图 S1）。</li>
		<li>单击 Add Primer to Template（将引物添加到模板）。</li>
	</ol></li>
</ol>3. PCR 扩增片段
<ol><li>设置六个单独的 PCR 反应（表 2）:对于片段 1，使用引物 P1 和 P2（参见 补充图 S2）;对于片段 2，使用引物 P3 和 P4（参见 补充图 S2）;对于片段 3，使用引物 P5 和 P6（参见 补充图 S2）;对于片段 4，使用引物 P7 和 P8（参见 补充图 S2）;对于片段 5，使用引物 P9 和 P10;并使用引物 P11 和 P12 扩增片段 6（参见 补充图 S2）。 注:使用前解冻、混合并短暂离心每种组分。</li>
	<li>使用 表 2 中的三步方案运行 PCR。</li>
	<li>向 5 μL PCR 产物中加入 1 μL 6x DNA 上样缓冲液，混合并短暂离心内容物。</li>
	<li>使用 1% 琼脂糖凝胶电泳分析样品。</li>
</ol>4. PCR 片段的纯化
注:使用凝胶提取试剂盒从凝胶中纯化 PCR 产物（参见 材料表）对于载体构建很重要。
<ol><li>向 45 μL PCR 产物中加入 9 μL 6x DNA 上样缓冲液，混合并短暂离心内容物。</li>
	<li>进行 1% 琼脂糖凝胶/goldview 电泳以分离 DNA 片段。 注:请勿重复使用 TAE 电泳缓冲液，因为其 pH 值会影响 DNA 片段的回收率。</li>
	<li>条带充分分离后，使用锋利的手术刀小心地切除 DNA 条带。 注:通过修剪掉多余的琼脂糖来最小化凝胶切片的大小。</li>
	<li>在干净的 1.5 mL 微量离心管中称取凝胶切片，向凝胶切片中加入等体积的结合缓冲液（例如，0.3 mL 至 0.3 g 切片），并在 60 °C 下孵育 7 分钟。</li>
	<li>将微型柱插入 2 mL 收集管中。将步骤 4.4 中的 700 μL DNA/琼脂糖溶液添加到迷你柱中。</li>
	<li>在室温下以 10,000 × g 离心 1 分钟。丢弃滤液并重复使用收集管。</li>
	<li>加入 300 μL 结合缓冲液，并在室温下以 13,000 × g 离心 1 分钟。丢弃滤液并重复使用收集管。</li>
	<li>加入 700 μL 洗涤缓冲液，并在室温下以 13,000 × g 离心 1 分钟。丢弃滤液并重复使用收集管。</li>
	<li>以最大速度旋转空迷你柱 2 分钟以干燥柱基质。将微型柱放入干净的 1.5 mL 微量离心管中。</li>
	<li>将 20 μL 去离子水直接添加到柱膜中心，并在室温下静置 2 分钟。以 13,000 × g 的速度离心 1 分钟。</li>
	<li>将 DNA 储存在 -20 °C。</li>
</ol>5. 线性化向量的制备
注意:制备质粒后，可以使用选定的酶对其进行切割。长消化或双酶消化对于确保所有 DNA 的消化至关重要。这将减少后续实验中假阳性克隆的数量。
<ol><li>pVAX1 线性化
	<ol><li>在室温下按所示顺序制备反应混合物（表 3）。 注:应添加水的体积以保持指示的总反应体积。在这里，用反应混合物中的酶消化 1 μg 质粒。根据质粒浓度，可以调节反应混合物中质粒的体积。</li>
		<li>轻轻混合;然后向下旋转。在 37 °C 下在加热块或水恒温器中孵育 60 分钟。</li>
		<li>使用凝胶提取试剂盒进行类似于第 4 节中 PCR 片段的纯化进行凝胶纯化（参见 材料表）。</li>
	</ol></li>
	<li>pVAX1-F1 线性化 注:pVAX1-F1 是从第一轮 pVAX1（NdeI 和 HindIII）和片段 1 重组中获得的载体。pVAX1-F2 是从 pVAX1-F1 （NdeI） 和片段 2 重组的轮次中获得的载体。pVAX1-F3 是从 pVAX1-F2 （NdeI） 和片段 3 重组的轮次中获得的载体。pVAX1-F4 是从 pVAX1-F3 （NdeI） 和片段 4 重组的回合中获得的载体。pVAX1-F5 是从 pVAX1-F4 轮（EcoRV 和 NtoI）和片段 5 重组中获得的载体。<ol><li>对于每种载体，按照所示顺序在室温下分别制备反应混合物（表 3）。</li>
		<li>轻轻混合;然后向下旋转。在 37 °C 下在加热块或水恒温器中孵育 60 分钟。</li>
		<li>使用凝胶提取试剂盒进行类似于第 4 节中 PCR 片段的纯化进行凝胶纯化（参见 材料表）。</li>
	</ol></li>
</ol>6. 亚克隆到新载体
注:使用 50-200 ng 载体和插入片段时，可以获得良好的克隆效率。
<ol><li>设置 ExonArt 无缝克隆和组装反应（表 4）。使用灭菌的去离子 H2O 将总反应体积调节至 10 μL 并混合。</li>
	<li>将反应物在热循环仪中于 50 °C 孵育 15-60 分钟。 将样品储存在冰上。 注意:将孵育时间延长至 60 分钟可以提高组装效率。</li>
	<li>在冰上解冻 DH5α 化学感受态细胞。</li>
	<li>向感受态细胞中加入 10 μL 组装产物;然后，通过上下吹打轻轻混匀。</li>
	<li>将混合物放在冰上 30 分钟。</li>
	<li>在 45 °C 下热休克 45 秒，然后将试管转移到冰上 3 分钟。</li>
	<li>向试管中加入 900 μL SOC 培养基。</li>
	<li>在 37 °C 振荡培养箱中以 225 rpm 摇动试管 1 小时。</li>
	<li>将转化反应物以 6,000 × g 离心 2 分钟。弃去上清液，将细胞重悬于 100 μL 新鲜 SOC 培养基中。</li>
	<li>将转化的细胞悬液铺在含有 50 μg/mL 卡那霉素的单独 LB 板上。</li>
	<li>将所有板在 37 °C 下孵育过夜。 从每个实验板中挑选单独的分离菌落。</li>
</ol>7. 分析转化体
<ol><li>将 8 个菌落挑取到 20 μL 含有 50 μg/mL 卡那霉素的 LB 培养基中。</li>
	<li>设置菌落 PCR 反应并运行 PCR（表 5）。 注:对于片段 1 的菌落 PCR 反应，使用引物 P1 和 P2（参见 补充文件 1）;对于片段 2，使用引物 P3 和 P4（参见 补充文件 1）;对于片段 3，使用引物 P5 和 P6（参见 补充文件 1）;对于片段 4，使用引物 P7 和 P8（参见 补充图 S2）;对于片段 5，使用引物 P9 和 P10;并使用引物 P11 和 P12 检测片段 6（参见 补充图 S2）。</li>
	<li>向 5 μL PCR 产物中加入 1 μL 6x DNA 上样缓冲液，混合并短暂离心内容物。</li>
	<li>使用 1% 琼脂糖凝胶电泳分析结果。</li>
	<li>在 5 mL 含有 50 μg/mL 卡那霉素的 LB 培养基中选择一个阳性菌落，并在 37 °C 下生长过夜。</li>
	<li>根据制造商的说明，使用质粒 DNA 迷你试剂盒（参见 材料表）获得质粒。</li>
	<li>使用 1% 琼脂糖凝胶电泳可视化结果。</li>
</ol>

在 pVAX1 表达载体中克隆全长 PRRSV 基因

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<li>Zhang, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+an+infectious+clone+of+HuN4-F112%2C+an+attenuated+live+vaccine+strain+of+porcine+reproductive+and+respiratory+syndrome+virus+high+copy+plasmid%5BTitle%5D)+AND+%22Virol+J%22%5BJournal%5D)">Generation of an infectious clone of HuN4-F112, an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus high copy plasmid.</a> Virol J. 8, 410(2011).</li>
<li>Ni, Y. Y., Huang, Y. W., Cao, D. J., Opriessnig, T., Meng, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+a+DNA-launched+infectious+clone+for+a+highly+pneumovirulent+strain+of+type+2+porcine+reproductive+and+respiratory+syndrome+virus%3A+identification+and+in+vitro+and+in+vivo+characterization+of+a+large+spontaneous+deletion+in+the+nsp2+region%5BTitle%5D)+AND+%22Virus+Res%22%5BJournal%5D)">Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region.</a> Virus Res. 160 (1-2), 264-273 (2011).</li>
</ol>

作者声明没有利益冲突。

Gibson 组装技术是一种基于 体外 重组的分子克隆方法，用于组装 DNA 片段8。该方法能够在单管等温反应中将多个 DNA 片段组装成一个环状质粒。然而，Gibson Assembly 技术的障碍之一是从 cDNA 获取长片段。由于多种原因，长片段难以准确扩增。例如，引物在长时间延伸时更容易错配，cDNA 质量可能不佳，或者富含 GC 的区域会阻止 DNA 聚合。当使用 PCR 无法从 cDNA 中轻易获得全长...

分子克隆是构建基于 DNA 的原核细胞和真核细胞表达实验工具的重要技术，是实验生物学中非常重要的组成部分。分子克隆涉及四个过程:获取插入片段 DNA、将插入片段连接到适当的载体中、将重组载体转化成大肠杆菌（大肠杆菌）以及鉴定阳性克隆1。到目前为止，已经采用了多种方法通过使用限制性内切酶 2,3 和 PCR 介导的重组 4,5,6 来连接 DNA 分子。同源重组，称为无缝克隆技术，是一组克隆方法，允许将一个或多个 DNA 片段不依赖于序列且无疤痕地插入载体中。该技术包括不依赖序列和连接的克隆 （SLIC）、无缝连接克隆提取物 （SLiCE）、In-Fusion 和 Gibson 组装。它采用核酸外切酶降解插入片段的一条链，利用载体生成内聚末端，并通过体内修复或体外重组通过形成磷酸二酯键将插入片段共价连接到载体上。将单个插入片段连接到任何序列的载体而没有任何疤痕的能力非常有吸引力。此外，该技术能够按预定顺序连接 5-10 个片段，而不受序列限制。
作为众多重组 DNA 技术之一，Gibson 组装技术是目前最有效的克隆方法 7,8，是一种强大而优雅的基于核酸外切酶的方法，可无缝组装一个或多个线性化 DNA 片段。Gibson 组装反应是在等温条件下使用三种酶的混合物进行的，即 5' 核酸外切酶、高保真聚合酶和热稳定 DNA 连接酶。由 5'-3' 核酸外切酶产生的单链 3' 突出端有助于在一端共享互补性的片段的退火。高保真聚合酶通过添加 dNTP 有效填充退火单链区域的间隙，热稳定的 DNA 连接酶密封缺口以形成关节 DNA 分子8。因此，该技术方法已被广泛用于基因表达载体的构建。
猪繁殖与呼吸综合征 （PRRS） 是一种病毒性疾病，可导致PRRSV 在任何 9 岁时引起猪的生殖障碍和呼吸衰竭。该综合征表现为发热、厌食、肺炎、嗜睡、抑郁和呼吸窘迫。此外，在一些流行病中观察到临床症状，包括耳朵的红色/蓝色变色。作为动脉病毒家族的一员，PRRSV 通过直接接触和交换液体（包括尿液、初乳和唾液）广泛传播到猪肉生产国。由于 PRRSV 在美国的传播，根据 580 万头母猪和 1.1 亿头猪的育种规模，猪肉行业的总经济损失估计约为每年 6.64 亿美元10,11。动植物卫生检验局报告显示，49.8% 的未接种疫苗的猪在血清12 中存在 PRRSV，受感染猪体内低水平的 PRRSV 通过唾液、鼻腔分泌物、尿液和粪便排泄13。已经实施了多种策略来控制 PRRSV 传播 14,15,16。除了消除程序以创建完全病毒阴性的群体或改善生物安全和管理外，接种疫苗是控制 PRRS 的有效手段。
PRRSV 是一种包膜、单链、正义 RNA 病毒，长度约为 15 kb。PRRSV 基因组由至少 10 个开放阅读框 （ORF）、一个短的 5' 非翻译区 （5' UTR） 和 3' 末端的 poly （A） 尾部组成（图 1A）17。负链 RNA 病毒的基因组是非传染性的，而正链 RNA 病毒的基因组是传染性的。生成病毒后代18 的 RNA 和 DNA 转染有两种主要策略。然而，克隆与 RNA 基因组相对应的全长片段对于感染性克隆的构建至关重要。由于 PRRSV 基因组的长而复杂，无法通过 PCR 一次轻松获得全长基因组。此外，尽管人工合成 PRRSV 基因是一种有效的解决方案，但长片段的合成通常很昂贵。因此，为了获得 PRRSV 全长表达载体，我们尝试通过多个插入片段同源重组方法创建它19,20。不幸的是，我们无法获得全长基因表达载体。因此，在本研究中，我们在反向引物中添加了合适的限制性位点，并通过几轮同源重组反应成功获得了 pVAX1-PRRSV 表达载体。此外，该方法还可以实现靶基因的缺失或突变，并有效地将大量 DNA 片段连接到表达载体上。

<table><tbody><tr><td>1 kb plus DNA Ladder</td><td>Tiangen Biochemical Technology (Beijing) Co., Ltd</td><td>MD113-02</td><td></td></tr><tr><td>2x Universal Green PCR Master Mix</td><td>Rong Wei Gene Biotechnology Co., Ltd</td><td>A303-1</td><td></td></tr><tr><td>Agarose</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>9012-36-6</td><td></td></tr><tr><td>Benchtop Microcentrifuge</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FRESCO17</td><td></td></tr><tr><td>Clean Bench</td><td>Sujing Antai Air Technology&amp;nbsp; Co., Ltd</td><td>VD-650-U</td><td></td></tr><tr><td>DNA Electrophoresis Equipment</td><td>Cleaver Scientific&amp;nbsp; Co., Ltd</td><td>170905117</td><td></td></tr><tr><td>DNA Loading Buffer (6x)</td><td>Biosharp Biotechnology Co., Ltd</td><td>BL532A</td><td></td></tr><tr><td>E. Z. N. A. Gel Extraction kit</td><td>Omega Bio-Tek Co., Ltd</td><td>D2500-01</td><td></td></tr><tr><td>E.Z.N.A. Plasmid DNA Mini Kit I</td><td>Omega Bio-Tek Co., Ltd</td><td>D6943-01</td><td></td></tr><tr><td>Electro-heating Standing-temperature Cultivator</td><td>Shanghai Hengyi Scientific Instrument Co., Ltd</td><td>DHP-9082</td><td></td></tr><tr><td>ExonArt Seamless Cloning and Assembly kit</td><td>Rong Wei Gene Biotechnology Co., Ltd</td><td>A101-02</td><td></td></tr><tr><td>ExonScript RT SuperMix with dsDNase</td><td>Rong Wei Gene Biotechnology Co., Ltd</td><td>A502-1</td><td></td></tr><tr><td>FastDigest Eco321 (EcoRV)</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0303</td><td></td></tr><tr><td>FastDigest HindIII</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0504</td><td></td></tr><tr><td>FastDigest NheI</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0974</td><td></td></tr><tr><td>FastDigest NotI</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0596</td><td></td></tr><tr><td>Gel Doc XR</td><td>Bio-Rad Laboratories Co., Ltd</td><td>721BR07925</td><td></td></tr><tr><td>Goldview Nucleic Acid Gel Stain</td><td>Shanghai Yubo Biotechnology Co., Ltd</td><td>YB10201ES03</td><td></td></tr><tr><td>Ice Maker Machine</td><td>Shanghai Bilang Instrument Manufacturing Co., Ltd</td><td>FMB100</td><td></td></tr><tr><td>Invitrogen Platinum SuperFi II DNA Polymerase</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>12361010</td><td></td></tr><tr><td>LB Agar Plate (Kanamycin)</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B530113-0010</td><td></td></tr><tr><td>LB sterile liquid medium (Kanamycin)</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B540113-0001</td><td></td></tr><tr><td>Micropipettors</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>&amp;mdash;</td><td></td></tr><tr><td>Microwave Oven</td><td>Panasonic Electric (China) Co., Ltd</td><td>NN-GM333W</td><td></td></tr><tr><td>Orbital Shakers</td><td>Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd</td><td>ZHWY-2102C</td><td></td></tr><tr><td>PRRSV virus</td><td>Sichuan Agricultural University</td><td>&amp;mdash;</td><td></td></tr><tr><td>SnapGene</td><td>GSL Biotech, LLC</td><td>v5.1</td><td>To design primers</td></tr><tr><td>T100 PCR Gradient Thermal Cycler</td><td>Bio-Rad Laboratories&amp;nbsp; Co., Ltd</td><td>T100 Thermal Cycler</td><td></td></tr><tr><td>TAE buffer</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B040123-0010</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>15596026</td><td>RNA extraction reagent</td></tr></tbody></table>

a seamless cloning approach for porcine reproductive and respiratory syndrome virus expression vector construction

基因表达载体的构建是实验生物学实验室工作的重要组成部分。随着 Gibson Assembly 等技术的进步，载体构建变得相对简单和高效。然而，当猪繁殖与呼吸综合征病毒 （PRRSV） 的全长基因组不能轻易地通过 cDNA 的单个聚合酶链反应 （PCR） 扩增，或者难以在 体外通过多个插入片段的同源重组获得全长基因表达载体时，目前的 Gibson Assembly 技术无法实现这一目标。
因此，我们的目标是将 PRRSV 基因组分成几个片段，并将适当的限制性位点引入反向引物以获得 PCR 扩增片段。通过同源重组技术将以前的 DNA 片段连接到载体中后，新载体获得了限制性内切酶切割位点。因此，我们可以使用新添加的酶切割位点对载体进行线性化，并在上游 DNA 片段的下游引入下一个 DNA 片段。
上游 DNA 片段 3' 端引入的限制性内切酶切割位点将被消除，新的切割位点将被引入下游 DNA 片段的 3' 端。通过这种方式，我们可以将 DNA 片段一一连接到载体上。该方法适用于成功构建 PRRSV 表达载体，是将大量片段组装到表达载体中的有效方法。

在本文中，我们提出了一种 体外 重组系统，该系统使用反向引物通过连续引入的限制性位点组装和修复重叠的 DNA 分子（图 1B）。该系统是一种简单高效的程序，包括制备线性载体和含有 PCR 引入的突出端的插入片段，引物具有适当的 5' 延伸序列和限制性位点; 体外 单一等温反应和重组产物向合适的宿主细菌的标准化学转化;以及选择阳性菌落并获得用于下一?...

作者聚焦:探索全长 DNA 片段的克隆技术

在这里，我们提出了一种通过在插入片段的 3' 末端引入合适的限制性位点来获得 pVAX1-PRRSV 表达载体的方案。我们可以通过同源重组技术将载体线性化，并将 DNA 片段一一连接到载体上。

猪繁殖与呼吸综合征病毒表达载体构建的无缝克隆方法

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson ...

该方案演示了冗长而复杂的基因表达载体的构建。当 cDNA 单次 PCR 无法获得全长 DNA 片段，或体外多次插入同源重组无法获得全长基因表达载体时，该方法适用。目前，使用多插入片段连接克隆来获得全长 DNA 片段，以便克隆到合适的载体中。该方法可实现靶基因的缺失或突变，并高效地将大量 DNA 片段连接到表达载体上。

a-seamless-cloning-approach-for-porcine-reproductive-respiratory

Research

JoVE Journal

Biology

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction

638 次观看.  China West Normal University. 该方案演示了冗长而复杂的基因表达载体的构建。当 cDNA 单次 PCR 无法获得全长 DNA 片段，或体外多次插入同源重组无法获得全长基因表达载体时，该方法适用。目前，使用多插入片段连接克隆来获得全长 DNA 片段，以便克隆到合适的载体中。该方法可实现靶基因的缺失或突变，并高效地将大量 DNA 片段连接到表达载体上。

视频: 作者聚焦:探索全长 DNA 片段的克隆技术

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

利用反向遗传学操作的裂谷热病毒MP - 12菌株，以提高疫苗的安全性和有效性的NSS基因

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

科研

免疫与感染

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

哺乳动物系统 CRISPR 指南 RNA 克隆

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

遗传学

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

呼吸系统同步病毒病毒特定核胶囊的生成和组装

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

生物化学

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

细菌人工染色体:对于正链RNA病毒研究的功能基因组学工具

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

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, ...

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

简化反向遗传学方法，恢复重组性轮状病毒表达报告蛋白

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...

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

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

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 ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...