| Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions
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Author:
Date:
2017-07-20
[Abstract] We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.
[摘要] 我们已经调整了CLIP-seq（交联 - 免疫沉淀和DNA测序）的方法来绘制与病毒粒子的蛋白质壳接触的壳化RNA片段。 关于接触病毒衣壳的RNA序列的方案报告的结果。
【背景】正义RNA病毒包括所有生命形式的病原体。具有二十面体形状的病毒具有病毒外壳蛋白在RNA基因组周围形成保护壳（Stockley等人，2013）。在噬菌体MS2和植物感染的Brome花叶病毒（BMV）中，外壳蛋白优先接触特异性RNA序列（Ni等人，2013; Hoover等人。 ，2016; Rolfsson等人，2016）。这些接触可以调节感染期间RNA释放的时间，病毒基因表达和病毒RNA复制（Hoover等人，2016）。鉴定衣壳RNA相互作用可以提供对病毒感染的规定的见解，并提供抑制病毒感染的手段。考虑到这一点，我们已经开发了一种方法，使用UV交联，RNA断裂，外壳蛋白的选择性沉淀和由RNA片段制备的cDNA的下一代测序来鉴定纯化的病毒体中的衣壳RNA接触。以下协议是针对BMV病毒粒子开发的。
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| Enzymatic Reactions and Detection of C3 Cleavage Fragments
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Author:
Date:
2014-08-20
[Abstract] The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 cleavage is in most cases mediated by an enzyme complex called the C3 convertase. However, it is now becoming increasingly clear that the cleavage of C3 by a range of ‘single’ proteases into bioactive C3a and C3b fragments is of high physiological significance. Here, we describe a protocol for the enzymatic cleavage of human C3 by the serine protease cathepsin L and the detection of the cleavage products C3a and C3b by western blotting as an ...
[摘要] 补体组分C3是补体系统的主要效应分子。 C3作为前酶在血液和间质液中循环，并通过酶裂解活化为C3a部分，用作化学引诱物和免疫细胞活化剂的经典过敏毒素和作为机体最有效调理素的C3b部分。 C3切割在大多数情况下由称为C3转化酶的酶复合物介导。 然而，现在越来越清楚的是，通过一系列"单一"蛋白酶将C3切割成生物活性C3a和C3b片段具有高生理学意义。 在这里，我们描述了通过丝氨酸蛋白酶组织蛋白酶L酶切割人C3的方案和通过蛋白质印迹检测裂解产物C3a和C3b作为这种酶反应的实例。
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