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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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+an+infectious+clone+of+HuN4-F112,+an+attenuated+live+vaccine+strain+of+porcine+reproductive+and+respiratory+syndrome+virus+high+copy+plasmid.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; Co., Ltd</td>
			<td>FRESCO17</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean Bench</td>
			<td>Sujing Antai Air Technology&#160; Co., Ltd</td>
			<td>VD-650-U</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Electrophoresis Equipment</td>
			<td>Cleaver Scientific&#160; 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&#160; Co., Ltd</td>
			<td>FD0303</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest HindIII</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0504</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest NheI</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0974</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest NotI</td>
			<td>Thermo Fisher Scientific&#160; 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&#160; 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&#160; Co., Ltd</td>
			<td>&#8212;</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>&#8212;</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&#160; 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&#160; 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。

As an essential technique to construct DNA-based experimental tools for expression in prokaryotic and eukaryotic cells, molecular cloning is a very important component of experimental biology. Molecular cloning involves four processes: the acquisition of insert DNA, ligation of the insert into the appropriate vector, transformation of the recombinant vector into Escherichia coli (E. coli), and identification of the positive clones1. So far, multiple methods have been adopted for joining DNA molecules by using restriction enzymes2,3 and PCR-mediated recombination4,5,6. Homologous recombination, known as seamless cloning technology, is the group of cloning methods, which allows sequence-independent and scarless insertion of one or more fragments of DNA into a vector. This technology includes sequence- and ligation-independent cloning (SLIC), Seamless Ligation Cloning Extract (SLiCE), In-Fusion, and Gibson Assembly. It employs an exonuclease to degrade one strand of the insert and a vector to generate cohesive ends, and either in vivo repair or in vitro recombination to covalently join the insert to the vector by forming phosphodiester bonds. The ability to join a single insert to a vector at any sequence without any scars is very appealing. Furthermore, the technology has the ability to join 5-10 fragments in a predetermined order without sequence restrictions.
As one of many recombinant DNA techniques, the Gibson Assembly technique, currently the most effective cloning method7,8, is a robust and elegant exonuclease-based method to assemble one or multiple linearized DNA fragments seamlessly. The Gibson Assembly reaction is performed under isothermal conditions using a mixture of three enzymes,namely, 5&#39; exonuclease, high-fidelity polymerase, and a thermostable DNA ligase. Single-strand 3&#180; overhangs created by the 5&#39;-3&#39; exonuclease contribute to the annealing of fragments that share complementarity at one end. The high-fidelity polymerase effectively fills the gaps in the annealed single-strand regions by adding dNTPs, and the thermostable DNA ligase seals the nicks to form joint DNA molecules8. Hence, this technical method has been widely used for the construction of gene expression vectors.
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that leads to reproductive impairment and respiratory failure in pigs caused by PRRSV at any age9. The syndrome is manifested as fever, anorexia, pneumonia, lethargy, depression, and respiratory distress. Moreover, clinical signs, including red/blue discoloration of the ears, have been observed in some epidemics. As a member of the family arterivirus, PRRSV is widely transmitted to pork-producing countries by direct contact and exchange of fluids, including urine, colostrum, and saliva. Due to the spread of PRRSV in the United States, the total economic losses of the pork industry have been estimated to be approximately $664 million per year, based on the breeding scale of 5.8 million sows and 110 million pigs10,11. The Animal and Plant Health Inspection Service report shows that 49.8% of unvaccinated pigs show the presence of PRRSV in serum12 and low levels of PRRSV in infected pigs are excreted through saliva, nasal secretions, urine, and feces13. Multiple strategies have been implemented to control PRRSV propagation14,15,16. In addition to elimination procedures to create completely virus-negative populations or improving biosafety and management, administering vaccines is an effective means of controlling PRRS.
PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a length of approximately 15 kilobases (kb). The PRRSV genome consists of at least 10 open reading frames (ORFs), a short 5&#39; untranslated region (5&#39; UTR), and a poly(A) tail at the 3&#39; terminus (Figure 1A)17. The genome of a negative-stranded RNA virus is non-infectious whereas the genome from positive-stranded RNA viruses is infectious. There are two main strategies for RNA and DNA transfection for generating virus progeny18. However, cloning the full-length fragment corresponding to the RNA genome is crucial for the construction of infectious clones. Due to the long and complex nature of the PRRSV genome, the full-length genome cannot be easily obtained through PCR at once. Additionally, although the artificial synthesis of PRRSV genes is an effective solution, the synthesis of long fragments is often expensive. Hence, to obtain the PRRSV full-length expression vector, we attempted to create it by the multiple inserts homologous recombination method19,20. Unfortunately, we were not able to obtain the full-length gene expression vector. Therefore, in this study, we added appropriate restriction sites to the reverse primer and successfully obtained the pVAX1-PRRSV expression vector by several rounds of homologous recombination reactions. Furthermore, this method can also achieve deletion or mutation of target genes and efficiently join a large number of DNA fragments to the expression vector.

作者聚焦：探索全长 DNA 片段的克隆技术

猪繁殖与呼吸综合征病毒表达载体构建的无缝克隆方法

This protocol demonstrates the construction of a lengthy and intricate gene expression vector. When a full-length DNA fragment cannot be obtained by a single PCR from cDNA, or the full-length gene expression vector cannot be obtained by multiple-insert homologous recombination in vitro, this method is applicable. Currently, multiple-insert ligation cloning is used to obtain full-length DNA fragment for cloning into a suitable vector.This method can achieve deletion or mutation of target genes and efficiently join a large number of DNA fragments to the expression vector.

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?...

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。

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

339 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

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal.
Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment.
The introduced restriction enzyme cleavage site at the 3&#39; end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3&#39; end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.

Here, we present a protocol to obtain the pVAX1-PRRSV expression vector by introducing suitable restriction sites at the 3' end of the inserts. We can linearize the vector and join DNA fragments to the vector one by one through homologous recombination technology.

科研

生物学

PCR Primer Design for Cloning of the Porcine Reproductive and Respiratory Syndrome Virus Gene

To begin, open the software and select the option to create a new DNA file. Prepare the PRRSV gene sequence from NCBI and click Okay to generate the sequence files. Analyze the sequence and mark the fragment junctions.Identify all the junctions before designing the specific sequence primers for the fragments. Then click on Primers and choose Add Primer. Paste the specific sequence and add overlap sequences of the vector to the first five prime nucleotide of the primer.Name the forward primer and click on Add Primer to Template to incorporate the designed primer into the template. Then mark the fragment junctions and design the reverse specific sequence primer of the fragments. Again, navigate to Primers and select Add Primer.Input the specific sequence. Add the restriction site to the first five prime nucleotide. Also, include 20 to 40 base pairs of vector overhang sequences to the first five prime nucleotide of the restriction site.Name the reverse primer and select Add Primer to Template. Next, set up six individual PCR reactions using the six fragments and designed primer pairs. Run the PCR following a three-step protocol as shown here.After PCR completion, pipette one microliter of six xDNA loading buffer with five microliters of the PCR product. Mix and centrifuge briefly. Analyze the samples using 1%agarose gel electrophoresis.To construct a full length gene, insert fragments of PRRSV were amplified using six PCR reactions. The sizes of the fragments were confirmed by agarose gel electrophoresis.

猪繁殖与呼吸综合征病毒基因克隆的 PCR 引物设计

Cloning of the Porcine Reproductive and Respiratory Syndrome Virus Gene into the pVAX1 Vector

Begin by linearizing the vector. Prepare the reaction mixture containing specific restriction enzymes at room temperature. Use a gel extraction kit to purify the linearized vectors, then incubate a 37 degrees Celsius for 60 minutes.Then, assemble the exon R seamless cloning and assembly reaction to sub-clone the PRRSV gene. Adjust the total reaction volume to 10 microliters with sterilized deionized water. Then, thoroughly mix.Incubate the mixture in a thermocycler for 15 to 60 minutes at 50 degrees Celsius. Afterwards, store the samples on ice. After transforming the vector into competent cells, pick eight colonies into 20 microliters of LB medium containing kanamycin.Set up a colony PCR reaction to analyze the transformants. A full length PRRSV over-expression vector was obtained by introducing PRRSV fragment one into the pVAX1 vector, creating pVAX1-F1. Successive rounds of recombination using specific restriction enzymes resulted in incorporation of fragments two through five, visualized by an increase in plasmid size.