Name: affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells
Uploaded: 2012-06-03T14:00:00
Description: Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

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...

<ol>
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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 ...

Research

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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 ...

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在鸡胚的流感病毒传播

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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 ...

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鼻湿巾从猪流感病毒检测和​​隔离

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