| Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks
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Author:
Date:
2016-05-20
[Abstract] Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., ...
[摘要] 呼吸道合胞病毒（RSV）属于包括许多临床相关病毒（例如人偏肺病毒和麻疹）的副粘病毒家族。 RSV感染可以在婴儿，老年人和一些免疫受损的成人中引起严重的疾病。在RSV复制期间，产生一系列截短形式的病毒基因组。这些截短的病毒基因组被称为缺陷型病毒基因组（DVG），并且由许多病毒产生（Lazzarini等人，1981; Rao和Huang，1982; Prince等人， ，1996; Sun等人，2015; Tapia等人，2013）。 DVG可以限制全长病毒的复制，并且是针对RSV的先天免疫应答的主要天然触发因子（Sun等人，2015; Tapia等人 ，2013）。在这里，我们详细讨论如何准备具有高或低含量的DVG的RSV股票，以及如何从富含缺陷病毒颗粒的RSV原液中纯化含有DVG的缺陷病毒颗粒。这些程序可用于制备实验室研究所需的病毒原液和有缺陷的病毒颗粒。简言之，在HEp2细胞中产生不同的RSV种群，其通常用于在实验室中扩增该病毒。为了产生具有高含量DVG的RSV原液，HEp2细胞用高多重感染（MOI）多次感染，然后使用梯度离心纯化含有DVG的病毒颗粒。这里描述的方法具有四个部分：1.具有低DVG含量（RSV-LD）的种子RSV原种的扩增，2.具有高DVG含量（RSV-HD）的原种的产生，3.通过梯度离心，4.纯化DVG的表征。
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| 3H-Penciclovir (3H-PCV) Uptake Assay
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Author:
Date:
2013-09-05
[Abstract] Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as 18F-FHBG and 124I-FIAU. 3H-Penciclovir (PCV) uptake assay is a method of choice used to determine the ...
[摘要] 来自人单纯疱疹病毒1型（HSV1-TK）的胸苷激酶与特定底物前药核苷酸类似物更昔洛韦（GCV）的组合已广泛用作癌症基因治疗的自杀性治疗基因。将具有改善的底物特异性的HSV1和其突变体（ HSV1-sr39TK ）用作小动物中各种生物学功能的PET成像的报告基因，通过与放射性标记的底物例如18 F-FHBG和124 I-FIAU。 H-Penciclovir（PCV）摄取试验是一种选择用于测定哺乳动物细胞和组织中HSV1-TK表达水平的方法。 HSV1-TK磷酸化PCV并导致形成penciclovir单磷酸，并且其随后通过细胞TK的磷酸化导致形成选择性地捕获在表达HSV-TK的细胞中的三磷酸喷昔洛韦。通过诸如闪烁计数的放射性方法，可以检测到更昔洛韦对哺乳动物细胞和组织的摄取。在这里我们描述在哺乳动物细胞中进行 H - Penciclovir摄取的方案。
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| Protein Translation Study – Label Protein with S35 Methionine in Cells
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Author:
Date:
2012-11-05
[Abstract] To follow protein synthesis, cells should be incubated with radioactive amino acid such as [35S] methionine during mRNA translation. Then, the neosynthetized protein will be identified by an autoradiography after immunoprecipitation with a specific antibody and separation on a polyacrylamide denaturing gel.
[摘要] 该实验方案的中文版正在准备中．．．
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