我们要感谢帕特里克·威尔逊的单克隆抗体CR9114。我们也感谢詹妮弗Debeauchamp和理查德·威比原A/Anhui/1/13质粒。由国立过敏规定这项工作的部分支持和传染病资助计划项目资助AI097092-01A1和CEIRS（卓越中心的流感研究和监测，HHSN26620070010C）。 FK是由来自奥地利科学基金会（FWF）的薛定谔奖学金（十3232）的支持。

1。重组杆状病毒的一代<ol><li>克隆到杆状病毒转移质粒<ol><li>克隆H7 HA和NA的N9胞外结构域（或任何其他HA或NA基因）插入修饰的pFastBac转移质粒（见介绍）。载体为含有C-末端三聚结构域和六组氨酸标记的HA，以及为NA含N-末端四聚结构域和六组氨酸标记，已经描述1,10,11,22（ 图1），并且可以从被请求的作者。 解释性说明：表示没有三聚化域医管局胞外结构域将被折叠不当，导致构象表位的中和损失，尤其是在茎部区域。同样，NA酶的功能依赖于正确的四聚。 </li><li>杆粒的产生 根据使用杆状病毒表达转换含有HA和NA基因导入DH10Bac感受态细菌杆状病毒转移质粒系统按照生产商的说明。隔离，并且使用按照制造商的说明（ 图2）一个Midiprep试剂盒纯化杆粒。 </li></ol></li><li>重组杆状病毒蛋白表达和验证的救援 解释性说明：下面的步骤必须在层流罩无菌条件下进行。独立执行此过程的HA和NA（或任何其他感兴趣的基因）。昆虫细胞通常生长在28°C，无二氧化碳 。 Sf9细胞对于此协议是生长在补充了10％FBS，1％青霉素-链霉素*和0.1％的Pluronic F68溶液，充分的TNM-FH昆虫细胞培养基，如果没有另外说明，并传代1点04分，每周两次。高五细胞生长在SFX无血清培养基传代1时15每周两次。 *添加抗生素应被视为可选的整个协议，并根据每个实验室中使用的做法调整。 <ol><li>板Sf9昆虫细胞中6孔板在2×10 5细胞/ cm 2的密度和让坐在在28℃培养箱中培养20分钟（不含CO 2）。 </li><li>拌2微克的重组杆状病毒质粒用100μl的TNM-FH培养基（无血清，1％青霉素 - 链霉素）中的（浓度0.5-2微克/微升）。搭配6微升转染试剂与100微升的TNM-FH培养基（无血清，青霉素 - 链霉素）的。结合两卷，轻轻混匀，让坐20分钟，在室温（转染混合物）。包括一个模拟转染对照（ 图3）。 </li><li>从接种Sf9细胞取出上清液（细胞现在坚持自己制版）和替换用2毫升含青霉素 - 链霉素（1％），血清游离TNM-FH培养基中。加入转染混合物的完整的体积（从步骤1.2.2）并轻轻摇动的6孔板中，持续5秒。孵育在28℃培养箱中无CO 2的6小时（ 图3）。 </li><li>更换介质无线个新鲜完整的TNM-FH培养基（含10％FBS，1％青霉素 - 链霉素，0.1％的Pluronic F68）。孵育在28℃培养箱中无CO 2的6天。检查偶见细胞在显微镜下。受感染的细胞通常会出现较大而圆，有细胞核增大，并开始分离（ 图4）。 </li><li>细胞和确认蛋白表达的收获<ol><li>收获细胞后第6天的转染通过离心在2000×g离心5分钟，在室温下进行。上清含有重组杆状病毒 - 这将被用于产生工作股马上，或可以被存储在4℃下多年。 </li><li>要检查蛋白表达收集颗粒重悬在500微升PBS。混合50微升悬浮液与50微升SDS-PAGE上样缓冲液（含有还原剂）。运行SDS-PAGE和用抗-六聚组氨酸抗体进行Western印迹分析来确认基因表达。包括从模拟囚室转染作为阴性对照（ 图3）。 </li></ol></li><li>工作个股的制备<ol><li>板5×10 5 Sf9细胞/厘米2的T175厘米2瓶，让坐20分钟让他们重视的菜。用含3％FBS（和1％青霉素 - 链霉素，0.1％的Pluronic F68）TNM-FH媒体取代完整的TNM-FH媒体。 </li><li>感染细胞用100微升救援上清液。孵育在28℃培养箱中无CO 2的6天，偶尔检查电池是否被感染（如步骤1.2.5中所述）。 </li><li>旋在2000×g离心，在室温下放置5分钟，并收集上清液。这是现在你的P2股票。这个股票可以无限期地在4°C下贮存用100微升的P2股票感染细胞重复感染程序。所得到的股票被称为P3或工作原液，它可以储存在4℃或直接用于蛋白质的表达。 P3的股票通常含有10 &lt;SUP&gt; 7 -10 9菌斑形成单位/毫升。 </li></ol></li></ol></li></ol> 2。蛋白质表达与纯化 解释性说明：Sf9细胞支持杆状病毒的成长非常好。然而，它们的分泌大量重组蛋白的能力是有限的。因此，我们使用另一种昆虫细胞系，击掌细胞，分泌的重组蛋白质的表达。这些细胞不支持杆状病毒复制到非常高滴度13，但具有较高的分泌能力。 <ol><li>长大后击掌细胞 长高五细胞SFX昆虫细胞培养液中T175厘米2瓶。通道每隔1:3一天。对于200毫升表达培养你将需要增长5汇合烧瓶。 </li><li>采收和感染击掌细胞<ol><li>通过敲击瓶用你的手从分离5 T175厘米2瓶击掌细胞。收集细胞suspension并转移到50ml离心管中。 </li><li>旋在1200×g下在室温下7分钟。用倾析法除去上清液，并通过他们再悬浮于15ml P3股票的团结颗粒。 </li><li>让在流罩坐15分钟（ 图5）。 </li><li>转移200毫升Hyclone公司SFX培养基倒入干净的，无菌千毫升摇瓶中（不含buffles，用高压灭菌前的铝箔密封的，不应该被用于细菌培养物之前）。 </li><li>转移P3/cell悬挂到摇瓶。轴封采用无菌铝箔和转移烧瓶成振动筛。摇在70转，28℃72-96小时。 </li></ol></li><li>从高五细胞表达上清收获重组蛋白<ol><li> 72-96小时后感染转让培养上清液到500ml离心桶。转速为5,500×g离心20分钟，在4°C。在此期间，冲洗摇瓶3倍用双蒸水（双蒸2 O）。 </li><li>转让3毫升镍树脂浆料倒入50毫升的试管用45毫升的PBS（一个需要每200毫升培养）的。摇匀和自旋在3000 XG在室温下10分钟。倒出PBS中，并保持颗粒。混合昆虫细胞上清与镍树脂颗粒，并转移到已经清洗摇瓶。孵育在4℃下2-4小时，同时振荡（ 图6）。 </li><li>上一个支撑架夹子山10毫升聚丙烯列。卸下两个盖子，并把该栏下方的烧杯中通（垃圾）收集流量。就拿摇瓶出振动筛，并开始上清/矿浆混合传输到列，它会保留镍树脂和只取上清液流过。通过整卷在列（ 图7）。 </li><li>用15ml洗涤缓冲液（50mM的Na 2 HCO 3，300 mM氯化钠，20 mM咪唑，pH 8）中洗涤树脂4倍。让所有的洗涤液漏从柱中，并与盖（下侧）将其关闭。 </li><li>加入2毫升洗脱缓冲液（50毫米的Na 2 HCO 3，300 mM氯化钠，300 mM咪唑，pH值8），并让坐5分钟。打开柱，收集洗脱液。重复此3次以上（含总共8毫升洗脱缓冲液）。洗脱液是含有高浓度蛋白质的通常显示时的泡沫振荡（ 图7）。不断洗出液对湿冰只要有可能。 </li></ol></li><li>缓冲液交换和浓度<ol><li>用PBS（pH值7.4-7.6），在3000 XG在4°C下20分钟Prespin15毫升超滤离心装置（视纯化蛋白大小30 kDa的截止用于HA / NA，但可能会有所不同）。丢弃流过负载和洗脱液到离心柱。用5ml无菌的，冰冷的PBS中，并旋为在3000×g离心60分钟，填充在4℃下</li><li>通过再次排出流，填充用15毫升冰冷的PBS及自旋为在3000×g离心60分钟，在4℃下重复此过程杜热一次。 </li><li>取离心柱出来的离心机，并收集交换缓冲液浓缩液（通常为200微升）。这是高度集中的重组蛋白质。冲洗离心柱用400微升无菌冰冷的PBS中并添加到精矿。保持该浓缩物在冰上在任何时候。 </li></ol></li></ol> 3。表征和质量控制<ol><li>测量蛋白质浓度 取浓缩液等分并使用你选择的蛋白质定量方法测量。通过加入PBS中设置的蛋白质浓度至1毫克/毫升，冷冻小等分试样至-80℃。冻融循环损害的蛋白质结构，并可能导致聚集。 </li><li> SDS-PAGE和考马斯亮蓝染色 运行500纳克的蛋白在SDS-PAGE和进行考马斯染色。快速和方便的染色使用考马斯亮蓝试剂结合微波协议。 HA蛋白通常出现在64kDa的，而NA蛋白应该运行在大约56 kDa的（ 图8A和B）的大小。使用所描述的协议，你应该不会看到任何降解产物。如果发生退化，通常表现在酵母/真菌污染的迹象。在这种情况下重复上述步骤，并确保保留一切无菌。 </li><li> Western blot检测/酶联免疫吸附 为了验证蛋白质身份，我们建议做Western印迹或ELISA测定与特定的抗体。理想情况下，酶联免疫吸附测定法是用其结合构象表位（ 例如，抗-HA抗体秆1）的抗体进行的。使用这样的抗体的蛋白质的正确折叠，可以证实（ 图8C）。 </li><li>测量NA活动 病毒NA的正确折叠，也可以通过测量NA活性的测定。我们建议您使用市售NA * STAR试验（只需按照用户指南）来执行此测试。 </li></ol>

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C., 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=Enhanced+immunogenicity+of+stabilized+trimeric+soluble+influenza+hemagglutinin.">Enhanced immunogenicity of stabilized trimeric soluble influenza hemagglutinin.</a> PLoS One. 5, (2010).</li><li>Dreyfus, C., 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=Highly+conserved+protective+epitopes+on+influenza+B+viruses.">Highly conserved protective epitopes on influenza B viruses.</a> Science. 337, 1343-1348 (2012).</li><li>Steel, J., 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=Live+attenuated+influenza+viruses+containing+NS1+truncations+as+vaccine+candidates+against+H5N1+highly+pathogenic+avian+influenza.">Live attenuated influenza viruses containing NS1 truncations as vaccine candidates against H5N1 highly pathogenic avian influenza.</a> J. Virol. 83, 1742-1753 (2009).</li><li>Fouchier, R. A., 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=Avian+influenza+A+virus+(H7N7)+associated+with+human+conjunctivitis+and+a+fatal+case+of+acute+respiratory+distress+syndrome.">Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 1356-1361 (2004).</li><li>Kost, T., Condreay, J., Jarvis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+as+versatile+vectors+for+protein+expression+in+insect+and+mammalian+cells.">Baculovirus as versatile vectors for protein expression in insect and mammalian cells.</a> Nat. Biotechnol. 23, 567-575 (2005).</li></ol>

作者什么都没有透露。

虽然使用杆状病毒表达系统在昆虫细胞中流感病毒HA和NA蛋白的表达通常是直接的，也有考虑几个重要的点。这一点很重要，因为在引进指出的，HA和NA的胞外结构域是，表达的融合与三聚或四聚域分别。这些结构域确保通常由跨膜结构域，这是不存在，如果只有胞外结构域被表达辅助分子的正确折叠。例如，在HA胞外域的无三聚化域的表达导致低聚和在茎结构域1的重要的中和表位的不稳定...

至于新型H7N9流感病毒株在中国最近出现的第一个反应，我们收购的质粒携带的血凝素（HA）和神经氨酸酶（NA）的前两个菌株的基因组序列。用这些质粒，我们能够在昆虫细胞中快速构建杆状病毒表达载体生产重组HA和NA的。该表达系统是公认的在我们的实验室，并已被证明举足轻重的我们很多正在进行的研究项目1-8。昆虫细胞能够正确折叠和翻译后修饰的蛋白质复合物。这些修饰包括N-连接的糖基化，这对许多病毒糖蛋白重要。因此，它是不足为奇的杆状病毒系统是相当既定用于表达流感病毒的表面抗原。事实上，许多的HA和NA的晶体结构已被使用昆虫细胞表达的蛋白9-11解决。的另一个优点系统掌握在HA / L的细胞培养（基于我们有超过50个不同的HA蛋白的经验）高达30毫克其可靠的高蛋白质产量 - 病毒感染驱动的表达从强多角体蛋白启动子的结果。 除去HA和NA蛋白的跨膜和endodomain的允许蛋白质分泌的，水溶性的版本的表达，从而极大地方便了纯化过程。此外，这些胞外结构域是六聚组氨酸标签，因此可以利用亲和层析纯化，用Ni 2 +的树脂进行纯化。 HA和NA蛋白的跨膜结构域有助于同源三聚体和同源四聚体的形成，分别。为了保存这些低聚，我们添加了一个C-端结构域三聚到HA-和N-端四聚功能域的NA构造（ 图1）。我们已经表明得出结论说，这样的三聚化域稳定功能，形成机制RVED医管局1的秆域的构象表位。对NA的正确四聚也可能有助于正确折叠和NA功能10。序列和杆状病毒转移载体窝藏三聚或四聚域可以从作者或在外地，9,10,12其他实验室要求。 对于分泌蛋白在昆虫细胞中表达的另一个重要问题是正确的细胞系的选择。虽然草地夜蛾源性Sf9细胞支持杆状病毒复制非常好，一般用于病毒拯救和繁殖，他们只有有限的容量分泌大量蛋白质。 粉纹夜蛾 BTI衍生-TN-5B1-4细胞（俗称高五）有较高的分泌能力而且是表达所选择的在本协议13,14的细胞系。此外，它是有用的，以减少或什至除去胎牛血清（FB从表达培养S）。因此，我们利用媒体与减少（3％）胎牛血清含量为越来越多的病毒工作的股票，我们在无血清培养基中进行表达。 重组HA和NA蛋白可用于许多免疫学技术。也许是最常见的一种是在酶联免疫吸附测定法用于在接种测定血清转换在人类或动物4,6,7,15。具有和NAS还用在ELISPOT分析既刺激分子和检测试剂为抗体分泌细胞16。它们作为分子毒饵使用允许具体排序为浆母或其他类型的B细胞，然后可以用来隔离（治疗）的单克隆抗体（mAb）。重组HA和NA分子被进一步用于这些单克隆抗体8的表征。其他的例子包括研究pH稳定性都有所不同病毒株明示或受体特异性，或定量评估特定酶NA活性或抗抑制剂。此外，重组的，纯化的HA可用于标准化的灭活流感病毒疫苗的HA含量。 重要的是，的rHA（并在一定程度上不适用）候选疫苗目前正在在动物模型和人体临床试验，其中一个候选疫苗正在2013年2,17-19许可被FDA测试。这些新颖的疫苗的优点是，由于这些蛋白质的重组体的性质，可避免产生的高生长重配病毒的劳动密集的过程。也许更加重要的是，他们的HA产量高，重复性好从应变应变。此外，因为这些疫苗都产的蛋的无系统，它们不含有蛋白质污染物，是有问题的鸡蛋过敏的个体。 在这里，我们选择了从小说中国H7N9流感病毒，A /映的两个菌株表达的HA和NA蛋白ui/1/13和A/Shanghai/1/13 20,21。这些是及时的例子，但是所描述的协议，可用于任何流感A和B的HA或NA蛋白的表达，并且可以适合于表达的任何其他分泌的病毒或细胞蛋白。三聚或四跨膜蛋白可以分别克隆到用于HA和NA的表达盒。然而，大多数可溶性分泌细胞蛋白，如干扰素例如，不需要为稳定一个额外的域，并且可以完全表达与C-末端六组氨酸标签。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus expression system</td>
			<td>Invitrogen</td>
			<td>10359-016</td>
			<td>Follow manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TNM-FH insect medium</td>
			<td>Gemini Bioproducts</td>
			<td>600-311</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone SFX-Insect</td>
			<td>Thermo Fisher</td>
			<td>SH30278.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellfectin II Reagent</td>
			<td>Invitrogen</td>
			<td>P/N58760</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F68</td>
			<td>Sigma</td>
			<td>1000702664</td>
			<td></td>
		</tr>
		<tr>
			<td>SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Qiagen</td>
			<td>1018240</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra Centrigual filters Ultracel 30K</td>
			<td>Millipore</td>
			<td>UFC902024</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Columns (5 ml)</td>
			<td>Qiagen</td>
			<td>34964</td>
			<td></td>
		</tr>
		<tr>
			<td>Max efficiency DH10Bac bacteria</td>
			<td>Invitrogen</td>
			<td>10361-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink HiPure Plasmid Filter Midiprep Kit</td>
			<td>Invitrogen</td>
			<td>K210015</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399-100G</td>
			<td>for elution and wash buffers</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>ATCC</td>
			<td><a href="http://www.atcc.org/Products/All/CRL-1711.aspx" target="_blank">CRL-1711</a></td>
			<td></td>
		</tr>
		<tr>
			<td>High Five cells</td>
			<td>Invitrogen</td>
			<td>B85502</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel h7n9 influenza virus using the baculovirus expression system

杆状病毒表达系统是一种强大的工具，用于重组蛋白的表达。在这里，我们用它来产生流感的正确折叠和糖基化版本的病毒表面糖蛋白 - 血凝素（HA）和神经氨酸酶（NA）。作为一个例子，我们选择了新的H7N9病毒，最近出现在中国所表达的HA和NA蛋白。然而，该协议可以很容易地适应任何其他甲型和乙型流感病毒株表达HA和NA蛋白。重组HA（的rHA）和NA（RNA）的蛋白质是免疫学测定法，例如酶联免疫斑点法和ELISA法重要的试剂，并且还广泛使用的疫苗标准化，抗体筛选，分离和表征。此外，重组NA分子可以用于筛选的小分子抑制剂，并用于表征与NA的酶的功能，以及其对抗病毒药物的敏感性是有用的。重组HA蛋白也蓓NG测试作为试验性疫苗在动物模型，并根据重组HA疫苗最近被FDA批准用于人体。我们在这里描述，以产生这些分子的方法是直线前进，并且可以在流感实验室研究方便，因为它允许产生大量的蛋白快速和低成本的。虽然这里我们专注于流感病毒表面糖蛋白，这种方法也可以用于生产其它病毒和细胞表面蛋白。

从A/Shanghai/1/13和A/Anhui/1/13 HA分子的表达以及与约20 mg / L的文化的产量。这两个菌株的NA分子呈中度表达水平，从0.2 A/Shanghai/1/13 mg / L的文化到0.7毫克/升A/Anhui/1/13。所有四种蛋白质制剂有很少到没有杂质（ 图8A和B）与已被纯度高于港人士可通过表达水平来解释。 A/Shanghai/1/13 HA用ELISA法与广谱中和茎-反应性人体单克隆抗体CR9114 23进一步分析。该mAb结合的茎部区域敏感...

Watch this Scientific Journal Video about Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System at J

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

在这里，我们描述来表达在昆虫细胞来源于小说中国H7N9病毒的正确折叠和功能的流感病毒表面抗原的方法。该技术可适于表达任何病毒或细胞表面蛋白的胞外结构域。

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

expression-functional-recombinant-hemagglutinin-neuraminidase

Research

JoVE Journal

Immunology and Infection

23.8K 次观看.  Icahn School of Medicine at Mount Sinai. 在这里，我们描述来表达在昆虫细胞来源于小说中国H7N9病毒的正确折叠和功能的流感病毒表面抗原的方法。该技术可适于表达任何病毒或细胞表面蛋白的胞外结构域。 

视频: Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

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

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

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 ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

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. ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...