| Isolation of Particles of Recombinant ASC and NLRP3
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Author:
Date:
2015-05-20
[Abstract] NLRP3 inflammasome is a multiprotein complex responsible for the activation of inflammatory caspase-1, resulting in processing and release of pro-inflammatory cytoquines IL-1β and IL-18 (Schroder and Tschopp, 2010). This inflammasome is composed of the sensor protein NLRP3 connected to caspase-1 through the adaptor protein ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) (Schroder and Tschopp, 2010). We and others have reported that upon inflammasome activation functional oligomeric inflammasome particles of NLRP3 and ASC were released from cells, acting as ...
[摘要] NLRP3炎症小体是负责炎症半胱天冬酶-1活化的多蛋白复合物，导致促炎细胞因子IL-1β和IL-18的加工和释放（Schroder和Tschopp，2010）。这种炎症小体由通过衔接蛋白ASC（凋亡相关的斑点样蛋白与半胱氨酸蛋白酶募集结构域）连接到caspase-1的传感蛋白NLRP3组成（Schroder和Tschopp，2010）。我们和其他人已经报道，在炎症小体激活时，NLRP3和ASC的功能性寡聚炎症小体颗粒从细胞中释放，充当危险信号，通过促进细胞外caspase-1的活化来扩增炎症（Baroja-Mazo等，
通过纯化ASC的重组颗粒或组成型激活的NLRP3突变体来研究寡聚ASC和NLRP3炎症小体颗粒的细胞外功能是可能的与相关的cryopyrin相关周期性综合征（CAPS，突变p.D303N），都标记有黄色荧光蛋白（YFP），并在HEK293细胞中表达。通过重组ASC或突变体NLRP3在HEK293细胞中的表达导致它们自发聚集成斑点（Baroja-Mazo等人，2014）的事实促进了纯化过程，并且方案最初从Fernandes-Alnemri和Alnemri（2008年）。
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| siRNA Screening for Genes Involved in HSV-1 Replication
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Author:
Date:
2014-08-20
[Abstract] Small interfering RNAs (siRNAs) are small (typically 18-24 nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As siRNAs are easy to use experimentally, and can be designed to target any gene (including pathogens), their use is perfectly suited to and easily adapted to high-throughput genome-wide screening methodologies and a range of phenotypic assays. Here we describe the use of a large siRNA library (>8,000 genes targeted ...
[摘要] 小干扰RNA（siRNA）是能够沉默转录后基因表达的小（通常为18-24个核苷酸）RNA分子，因此，它们提供了一种简单的方法，通过其可以容易地评估单个基因在复杂细胞系统中的作用。 由于siRNA易于实验使用，并且可以设计为靶向任何基因（包括病原体），它们的使用完全适合于并且容易地适应于高通量全基因组筛选方法学和一系列表型测定。 这里我们描述了使用大的siRNA文库（> 8,000个基因单独靶向）来筛选和鉴定功能上涉及人疱疹病毒（1型单纯疱疹病毒; HSV-1）（Griffiths）的复制的宿主因子， et al。，2013; Griffiths，2013）。
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| VZV Replication Assays
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Author:
Date:
2014-08-20
[Abstract] Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency (von Bokay, 1909). Whilst VZV is extensively studied, inherent features of VZV replication, such as cell-association of virus particles during in vitro culture and a restricted host range (limited to humans and some other primates) mean the cellular and viral mechanisms underlying VZV reactivation and pathogenesis remain largely uncharacterised. Much remains to be learnt about VZV, interactions with its host, and the ...
[摘要] 水痘带状疱疹病毒（VZV）是人疱疹病毒，其在初次感染时引起水痘（水痘），在从潜伏期再活化后引起带状疱疹（带状疱疹）（von Bokay，1909）。虽然VZV被广泛研究，VZV复制的固有特征，例如在体外培养期间病毒颗粒的细胞缔合和受限的宿主范围（限于人和一些其它灵长类动物）意味着细胞和病毒基本的VZV再激活和发病机制仍然很大程度上没有表征。关于VZV，与其宿主的相互作用和疾病的发展仍然有很多知识。该协议描述了使用重组VZV-GFP报道病毒的基本VZV复制测定。由于VZV在组织培养中是高度细胞相关的，这里描述的报道病毒接种物是感染细胞的制备物。这种报道病毒感染的细胞系可以与siRNA基因缺失或cDNA过表达转染方案组合使用，以确定单个细胞基因对病毒复制的影响。
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