作者要感谢为rWSN PB2-链球菌病毒纳达Naffakh和玛丽 - 安妮Rameix Welti（巴斯德研究所）。

<html>一个协议的原理流程图如图。 1和试剂表呈列如下。 1。感染（16 - 24小时） <ol><li>出HeLa细胞在5 150毫米塑料杜尔贝科的改良Eagle培养基（DMEM培养基），高糖培养基，这样，他们将达到约80％汇合翌日（约5×10 8个细胞）细胞在培养皿中的种子。重要的是，这些细胞没有达到100％汇合。 </li><li>第二天，稀释到的链球菌标签的流感病毒库存磷酸盐缓冲液（PBS）含0.3％牛血清白蛋白（BSA）的多重感染3。 </li><li>从细胞生长培养基，用PBS洗一次，添加10毫升PBS含有病毒的细胞。在室温下1小时的孵育。 </li><li>更换培养液高糖感染的媒介，继续孵化，在37°C。 </li></ol>“&gt;的潜伏期长短取决于感染的研究阶段（见讨论）。 2。亚细胞分离（3小时） <ol><li>一次用冷PBS洗涤细胞和暂停冷PBS用橡皮刮刀和转让到50毫升锥形管细胞。 </li><li>颗粒细胞在表顶部的离心5分钟1000转，然后吸的PBS。 </li><li>重悬细胞沉淀在10毫升冷蔗糖缓冲（10毫米肝素钠pH值7.9，氯化钾10毫米，2毫米镁醋酸（MgOAc），3毫米氯化钙 ，蔗糖340毫米，1毫米二硫苏糖醇（DTT），1％的蛋白酶抑制剂混合） 。暂停后，通过细胞200μl的吸管尖连接到一个10毫升的塑料血清移液管，破坏细胞团块。 </li><li>在冰上孵育10分钟。轻轻颠倒定期悬浮细胞。 </li><li> Nonidet的P-40（NP-40）在高速的终浓度为15秒到0.5％和旋涡。立即在4°C离心10分钟在3500 XG表顶部的离心GE。 </li><li>删除上清并存储在4°C。这是细胞质的一部分。颗粒要小，比原来的细胞沉淀白。所有的分数可以在4°C保存至少4小时</li><li>洗5毫升冷蔗糖缓冲液，再离心沉淀在步骤2.5和弃上清。 </li><li>悬浮颗粒（含核）1.5毫升冷核质提取缓冲液（50 mM的肝素钠pH值7.9，150毫米KOAc，1.5毫米氯化镁 ，0.1％NP-40，数码地面1毫米，1％蛋白酶抑制剂组合）。 </li><li>转移到冷藏，全玻璃4毫升Dounce匀浆紧身杵和20杆，均质细胞核。 在同质化，避免起泡。阻力后，应增加约10招。相衬显微镜下可以确认有效的同质化。 </li><li>均质核转移到1.5毫升离心管和孵化一个旋转的轮子上20分钟，在4°C。 </li><li>在离心10分钟，在4°C离心​​16,000 xĞ。 </li><li>删除上清并存储在4°C。这是核质的分数。 </li><li>重悬浮颗粒在1.5毫升的消化缓冲液（50 mM的肝素钠pH值7.9，10 mM氯化钠， 氯化镁 1.5毫米，1毫米数码地面电视，1％的蛋白酶抑制剂的组合，100单位/毫升Benzonase（RNA分析，20 U / ml的酶代替无DNA酶I））预先加热至37℃，孵育10分钟，在37°C。 </li><li>加入42微升5 M氯化钠（终浓度为150 mM氯化钠）和孵育20分钟，在4°C。 </li><li>在离心10分钟，在4°C离心​​16,000 xĞ。 </li><li>删除上清并存储在4°C。这是的ch150分数。 </li><li>沉淀重悬在1.5毫升冷高盐缓冲液（50毫米肝素钠pH值7.9，500 mM氯化钠，MgCl 2的 1.5毫米，0.1％NP-40，1毫米数码地面电视，1％蛋白酶抑制剂组合）和孵育20分钟冰。 </li><li>在16000离心x G &lt;/ EM&gt;在在4°C离心10分钟。 </li><li>删除上清并存储在4°C。这是的ch500分数。 </li></ol> 3。链球菌标记净化（2小时） <ol><li>加入300μl5 M氯化钠的细胞质部分（终浓度为150 mM氯化钠）。 </li><li>等分包装链球菌Tactin琼脂糖珠每分数在15毫升锥形管100微升。 </li><li>倒置管，珠链球菌洗涤缓冲液加入10卷（20毫米肝素钠pH值7.9，150 mM氯化钠（500 mM氯化钠为ch500样品），1毫米EDTA）混合，离心5分钟，在1000×Ğ在桌面离心。 </li><li>弃上清，重复步骤3.3的两倍，然后悬浮在珠链球菌洗涤缓冲液等体积​​的颗粒。 </li><li>加入200μL洗链球菌Tactin珠浆每个分数为nuceloplasmic 1.5毫升离心管，ch150，ch500分数和细胞质分数15毫升锥形管。 </li><li>轮流管1H在4°Ç一个旋转的轮子上。 </li><li>离心管1 4分钟1000×Ğ°C。弃上清。 </li><li>每管加入1毫升的冷链球菌洗涤缓冲。转珠管（胞质分数）从15毫升到1.5毫升管。 4管1分钟°C间，在步骤3.7离心后洗净。重复此清洗步骤两次。 </li><li>最后的洗涤步骤后，洗涤缓冲液中删除所有的痕迹，使用一个单位的枪头。加入100μL洗脱缓冲液（链球菌洗涤缓冲液2.5毫米desthiobiotin），轻轻混匀。 </li><li>在冰上孵育15分钟。轻轻混珠偶尔轻敲管底部。 </li><li>离心管1000×Ğ1分钟在4°C的离心。收集上清含洗脱链球菌标记vRNPs的的。 </li></ol> 4。代表结果分馏的效率是最好的分馏裂解免疫印迹使用antib分析的判断odies特定的亚细胞标记（ 图2）。具体来说，成功的亚核分馏应该表现出的核质很少或没有细胞的RNA聚合酶II（聚合酶II），或可溶性核分数，最应提取150毫米氯化钠5。聚合酶II退化也再现观察流感病毒感染的细胞，而未受感染的细胞，与文献6一致。 vRNPs净化是最好的判断银或考马斯染色，在图细胞质洗脱液所示。 3。在我们的经验，最链球菌PB2纯化的完全形成vRNP的一部分，即微溶于聚合酶捕获。这反映在高NP比：PA/PB1/PB2的比例由银或考马斯染色观察。较低的比率表明核糖核酸酶缓冲污染，几个无处不在的核糖核酸酶（如RNA酶A），以解离这样的方式被称为裂解病毒RNA吃了聚合酶的NP多聚体7。有趣的是，Benzonase单独处理，同时充分消化病毒RNA，似乎没有任何效果stochiometric比率vRNP蛋白质。虽然我们无法解释这种现象，我们观察到中断后与其他核糖核酸酶（数据未显示）表明Benzonase，消化vRNPs可能会留下一定的结构RNA的重要地区保持不变。 vRNPs细胞质和核质（无核酸的消化执行）净化后，8淡淡的，但离散带，分子量高，可以观察到银染，这对应于8个流感基因片段的预测分子量（见文献3 ）。 如图从每个部分纯化vRNPs在9小时后感染的相对含量。 4。的vRNPs分布因在感染过程中，最强的在ch150分数积累在早期的时间点，在年底的时间点，增加积累，在胞核和胞浆。 <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="图1"/> 图1。亚细胞分离流程图。改编自文献。 3。 <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="图2"/> 图2。西方blot分析流感病毒感染的细胞，亚细胞组分。 9小时之前，亚细胞分离流感病毒株的WSN 10 9 HeLa细胞感染。等量的总蛋白被装在每个通道使用的抗体显示和分析。 RNA聚合酶II（聚合酶II）的检测，使用克隆8WG16，承认所有形式的C-端结构域（的hypophosphorylated带最为突出）。改编自文献。 3。 <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="图3"/> 图3。银染色分析纯化vRNPs的。 HeLa细胞感染rWSN（阴性对照）或9小时的rWSN-PB2-链球菌，链球菌净化从细胞质分数。星号表示带RNase消化后，这是不可见的。改编自文献。 3。 <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="图4"/> 图4。银污渍分析，从不同的细胞组分纯化vRNPs。 10 9 HeLa细胞感染rWSN PB2-链球菌或rWSN的9小时其次是亚细胞分离和纯化链球菌。从各部分洗脱液等量加载。改编自文献。 3。

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没有利益冲突的声明。

虽然最近已经有很多研究发现流感病毒感染的8所涉及的个别蛋白质或蜂窝网络，大多数这些相互作用的功能意义尚不清楚。鉴于流感病毒RNA的合成和复杂的生物物理和生物化学性质的核9基于染色质功能的绝对依赖，新技术将被要求澄清这些功能。我们在座的亚核分馏，加上亲和纯化的vRNPs，允许表征至关重要病毒RNA工厂这是以前无法进入的。 许多亚细胞分馏...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液，高血糖</td><td> Gibco公司</td><td> 11965-092 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A9418 </td><td></td></tr><tr><td>蛋白酶抑制剂混合Ğ </td><td> SERVA </td><td> 39101 </td><td></td></tr><tr><td> benzonase核酸</td><td> Novagen公司</td><td> 71206 </td><td> 25 U /μL </td></tr><tr><td>无RNase的DNase I可</td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U /μL </td></tr><tr><td> Dounce匀浆</td><td>惠顿</td><td> 432-1271 </td><td>使用类型“B”杵</td></tr><tr><td>链球菌-Tactin琼脂糖凝胶</td><td> IBA的有限责任公司</td><td> 2-1201-025 </td><td>也可以用50％的悬浮列格式</td></tr><tr><td> desthiobiotin </td><td>国际律师协会有限责任公司</td><td> 2-1000-002 </td><td></td></tr></tbody></table>

affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

像所有的负链RNA病毒，流感病毒的基因组在病毒核蛋白复合物（vRNP），单链的基因组，其中encapsidated由核蛋白（NP），三聚聚合酶组成的复杂关联的形式打包的PA，PB1和PB2亚基。然而，在大多数RNA病毒，流感病毒感染细胞的细胞核中执行病毒RNA的合成。有趣的是，病毒mRNA的合成采用细胞mRNA前，为引物，并已提出，这个过程需要1对染色体的地方。也被病毒聚合酶和RNA聚合酶II的主机，以及NP和主机的核小体之间的相互作用特点1,2。 近日，代编码单链球菌标签的重组流感病毒的基因融合病毒聚合酶PB2亚基C-末端（rWSN铅，链球菌3）已成为EN描述。这些重组病毒PB2-含复合物，包括vRNPs从被感染的细胞，使净化。为了获得纯化vRNPs，被感染的细胞培养，vRNPs有亲和力，从来自这些细胞裂解纯化。然而，迄今用于裂解程序已根据上一步的洗涤剂溶解，尽管一般核酸的存在，往往只提取染色质结合材料低效。 我们的前期工作的建议，没有在传统的细胞裂解提取大量的核vRNPs的部分，因此，不能亲和纯化。为了提高提取效率，并从非绑定到染色质的核vRNPs分开染色绑定，我们适应了一步明智的亚流感病毒感染的细胞提取协议。简言之，此过程首先从细胞核分开，然后提取可溶性核蛋白（这里称为“核质”的部分）。其余的不溶性的核材料，然后消化Benzonase，非特异性的DNA / RNA核酸，其次是两个盐提取步骤：首先使用150 mM氯化钠（称为“ch150”），然后500毫米的NaCl（“ch500”（ 图1） ）。根据我们的观察，这些盐的提取步骤被选为500 mM氯化钠充分溶解超过核vRNPs 85％，但仍允许亲和力矩阵标签vRNPs具有约束力。 受感染的细胞的亚细胞分离后，它是可能亲和力净化PB2标签从每个人的分数vRNPs和分析其蛋白质和RNA的组成部分，分别用Western blot和引物延伸，。最近，我们利用这种方法，发现在感染后的后期染色质部分提取500 mM氯化钠（ch500）3点，vRNP出口复合物的形式。

Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

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

流感病毒与宿主细胞染色体复制他们在协会的RNA基因组。在这里，我们提出了一个方法来净化完整的病毒核蛋白复合物，从感染细胞的染色质。纯化病毒复合物，可以分析，Western blot和引物蛋白质和RNA含量的扩展，分别。

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 ...

affinity-purification-influenza-virus-ribonucleoprotein-complexes

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Immunology and Infection

13.0K 次观看.  Universitätsklinikum Freiburg. 流感病毒与宿主细胞染色体复制他们在协会的RNA基因组。在这里，我们提出了一个方法来净化完整的病毒核蛋白复合物，从感染细胞的染色质。纯化病毒复合物，可以分析，Western blot和引物蛋白质和RNA含量的扩展，分别。

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

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

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

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

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

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 ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

生物膜启动对病毒感染的细胞测定细菌和真菌

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...