Name: expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel h7n9 influenza virus using the baculovirus expression system
Uploaded: 2013-11-06T23:00:00
Description: 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

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?...

<ol>
	<li>Krammer, F., 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=A+carboxy-terminal+trimerization+domain+stabilizes+conformational+epitopes+on+the+stalk+domain+of+soluble+recombinant+hemagglutinin+substrates.">A carboxy-terminal trimerization domain stabilizes conformational epitopes on the stalk domain of soluble recombinant hemagglutinin substrates.</a> PLoS One. 7, e43603 (2012).</li><li>Krammer, F., Pica, N., Hai, R., Margine, I., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+hemagglutinin+influenza+virus+vaccine+constructs+elicit+broadly-protective+stalk-specific+antibodies.">Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly-protective stalk-specific antibodies.</a> J. Virol. , (2013).</li><li>Leyva-Grado, V. H., Mubareka, S., Krammer, F., C&#225;rdenas, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+infection+in+Guinea+pigs+raised+as+livestock,+ecuador.">Influenza virus infection in Guinea pigs raised as livestock, ecuador.</a> Emerg. Infect. Dis. 18, 1135-1138 (2012).</li><li>Margine, I., 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=H3N2+influenza+virus+infection+induces+broadly+reactive+hemagglutinin+stalk+antibodies+in+humans+and+mice.">H3N2 influenza virus infection induces broadly reactive hemagglutinin stalk antibodies in humans and mice.</a> J. Virol. , (2013).</li><li>Miller, M. S., 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=1976+and+2009+H1N1+Influenza+Virus+Vaccines+Boost+Anti-Hemagglutinin+Stalk+Antibodies+in+Humans.">1976 and 2009 H1N1 Influenza Virus Vaccines Boost Anti-Hemagglutinin Stalk Antibodies in Humans.</a> J. Infect. Dis. 652, (1976).</li><li>Pica, N., 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=Hemagglutinin+stalk+antibodies+elicited+by+the+2009+pandemic+influenza+virus+as+a+mechanism+for+the+extinction+of+seasonal+H1N1+viruses.">Hemagglutinin stalk antibodies elicited by the 2009 pandemic influenza virus as a mechanism for the extinction of seasonal H1N1 viruses.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 2573-2578 (2012).</li><li>Sangster, M. Y., 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=The+B+cell+response+and+hemagglutinin+stalk-reactive+antibody+production+in+different+age+cohorts+following+2009+H1N1+influenza+vaccination.">The B cell response and hemagglutinin stalk-reactive antibody production in different age cohorts following 2009 H1N1 influenza vaccination.</a> Clin. Vaccine Immunol. , (2013).</li><li>Tan, G. S., 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=A+pan-h1+anti-hemagglutinin+monoclonal+antibody+with+potent+broad-spectrum+efficacy+in+vivo.">A pan-h1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo.</a> J. Virol. 86, 6179-6188 (2012).</li><li>Ekiert, D. 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=Antibody+recognition+of+a+highly+conserved+influenza+virus+epitope.">Antibody recognition of a highly conserved influenza virus epitope.</a> Science. 324, 246-251 (2009).</li><li>Xu, X., Zhu, X., Dwek, R. A., Stevens, J., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+the+1918+influenza+virus+H1N1+neuraminidase.">Structural characterization of the 1918 influenza virus H1N1 neuraminidase.</a> J. Virol. 82, 10493-10501 (2008).</li><li>Stevens, 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=Structure+of+the+uncleaved+human+H1+hemagglutinin+from+the+extinct+1918+influenza+virus.">Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus.</a> Science. 303, 1866-1870 (2004).</li><li>Wei, C. 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=Comparative+efficacy+of+neutralizing+antibodies+elicited+by+recombinant+hemagglutinin+proteins+from+avian+H5N1+influenza+virus.">Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus.</a> J. Virol. 82, 6200-6208 (2008).</li><li>Krammer, F., 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=Trichoplusia+ni+cells+(High+Five)+are+highly+efficient+for+the+production+of+influenza+A+virus-like+particles:+a+comparison+of+two+insect+cell+lines+as+production+platforms+for+influenza+vaccines.">Trichoplusia ni cells (High Five) are highly efficient for the production of influenza A virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines.</a> Mol. Biotechnol. 45, 226-234 (2010).</li><li>Palmberger, D., 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=Insect+cells+for+antibody+production:+evaluation+of+an+efficient+alternative.">Insect cells for antibody production: evaluation of an efficient alternative.</a> J. Biotechnol. 153, 160-166 (2011).</li><li>Santiago, F. W., Lambert Emo, K., Fitzgerald, T., Treanor, J. J., Topham, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+and+immunogenic+properties+of+recombinant+hemagglutinin+proteins+from+H1N1+A/Brisbane/59/07+and+B/Florida/04/06+when+produced+in+various+protein+expression+systems.">Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems.</a> Vaccine. 30, 4606-4616 (2012).</li><li>Wrammert, 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=Broadly+cross-reactive+antibodies+dominate+the+human+B+cell+response+against+2009+pandemic+H1N1+influenza+virus+infection.">Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection.</a> J. Exp. Med. 208, 181-193 (2011).</li><li>Treanor, J. 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=Protective+efficacy+of+a+trivalent+recombinant+hemagglutinin+protein+vaccine+(FluBlok)+against+influenza+in+healthy+adults:+a+randomized,+placebo-controlled+trial.">Protective efficacy of a trivalent recombinant hemagglutinin protein vaccine (FluBlok) against influenza in healthy adults: a randomized, placebo-controlled trial.</a> Vaccine. 29, 7733-7739 (2011).</li><li>Dalakouras, T., Smith, B., Platis, D., Cox, M., Labrou, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+recombinant+protein-based+influenza+vaccine.+Expression+and+affinity+purification+of+H1N1+influenza+virus+neuraminidase.">Development of recombinant protein-based influenza vaccine. Expression and affinity purification of H1N1 influenza virus neuraminidase.</a> J. Chromatogr. A. 1136, 48-56 (2006).</li><li>Krammer, F., Grabherr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+influenza+vaccines+made+by+insect+cells.">Alternative influenza vaccines made by insect cells.</a> Trends Mol. Med. 16, 313-320 (2010).</li><li>Gao, 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=Human+Infection+with+a+Novel+Avian-Origin+Influenza+A+(H7N9)+Virus.">Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus.</a> N. Engl. J. Med. , (2013).</li><li>Goff, P. H., 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=Induction+of+cross-reactive+antibodies+to+novel+H7N9+influenza+virus+by+recombinant+Newcastle+disease+virus+expressing+a+North+American+lineage+H7+subtype+hemagglutinin.">Induction of cross-reactive antibodies to novel H7N9 influenza virus by recombinant Newcastle disease virus expressing a North American lineage H7 subtype hemagglutinin.</a> J. Virol. , (2013).</li><li>Weldon, W. 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>

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 ...

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

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

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

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

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

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

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

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

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

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

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

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 ...