| Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells
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Author:
Date:
2020-04-05
[Abstract] A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus “piggy-backed” on T4 capsid, by virtue of its natural ability to enter many types of human cells ...
[摘要] [摘要 ] 一种病毒载体，可以安全有效地将大量多样的分子货物运送到细胞中 是治愈许多人类疾病的圣杯。腺伴随病毒（AAV）已被广泛使用，但容量很小。T4原核病毒容量大，但缺乏进入哺乳动物细胞的天然机制。在这里，我们通过将T4和AAV结合到一个具有两者优势的纳米颗粒中，创建了一种杂交载体。使用噬菌体修饰蛋白Soc（小的外衣壳蛋白）和Hoc（高度抗原化的外衣壳蛋白），通过亲和素-生物素交叉桥将25 nm的AAV小颗粒连接到120 nm x 86 nm的大T4头上。因此，AAV凭借其固有的进入多种类型人体细胞的自然能力，可以“背负”于T4衣壳上，从而有效地充当了“驱动器”，以运送与T4头相关的大型货物。这种独特的T4-AAV杂交载体方法可为将来开发新型疗法铺平道路。
[背景 ] 已经有新的和有效的递送载体能够运输基因和蛋白质的大货物进入人类细胞，以刺激生产治疗性生物分子的和/或修复的细胞和遗传缺陷的迫切需要。这样的载体将允许将快速出现的技术（例如CRISPR，CAR T细胞等）转化为用于大规模应用以及个性化医学的疗法（Stewart 等，2016）。
将具有不同特性的纳米粒子组装到杂化复合物中是开发新型功能材料的有力策略，因为这些杂化复合物显示出集体和协作的属性，其中某些属性可能与单个粒子所显示的属性不同（Ghosh 等人，2012； ...
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| Expression and Purification of Adeno-associated Virus Virus-like Particles in a Baculovirus System and AAVR Ectodomain Constructs in E. coli
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Author:
Date:
2020-02-05
[Abstract] Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 (AAV-2) and (b) two fragments from the binding ectodomain of AAV’s cellular receptor, AAVR. His6PKD1-2 comprises the first two polycystic kidney disease (PKD) domains, the minimal required for efficient binding of AAV, expressed with an N-terminal histidine tag. MBP-PKD1-5 is a fusion of the maltose binding protein with all five of the PKD domains of the AAVR receptor. Presented are the expression and purification of milligram quantities, ...
[摘要] [摘要] 腺伴随病毒（AAV）是一种有前途的基因治疗载体，其与宿主蛋白相互作用的生物物理特征是工程靶向组织和免疫逃逸的关键基础。这里介绍的是生产的协议：（a）血清型2（AAV-2）的外壳蛋白壳（病毒样颗粒或VLP），以及（b）来自AAV细胞受体AAVR结合胞外域的两个片段。他的6个PKD1-2包含前两个多囊肾疾病（PKD）域，这是有效结合AAV所需的最低限，以N端组氨酸标签表达。MBP-PKD1-5是麦芽糖结合蛋白与AAVR受体的所有五个PKD域的融合体。提出了毫克量的表达和纯化方法，足以进行体外分析。对于AAV-2，该协议为使用（传染性）野生型病毒或转导载体提供了一种替代方法。产生转导载体的方法之一是在Sf9细胞中，并且VLP的产生是基于此。对于AAVR，该协议可以对病毒结合进行生化和生物物理表征。最小的两个结构域的构建体可以更饱和地结合到病毒上的对称等效位点，而更大的构建体可能更好地反映了天然受体。
[背景 ] 的人细小病毒腺相关病毒（AAV）具有60亚基蛋白衣壳壳含有单链DNA基因组（解等人，2002 ; 查普曼和Agbandje-McKenna的2006）。在新兴的临床基因治疗应用中，几种重组AAV血清型被用作载体（Gaudet 等，2013；Russell ...
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| Co-sedimentation Assay for the Detection of Direct Binding to F-actin
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Author:
Date:
2012-10-05
[Abstract] This protocol describes measurement of direct protein-protein interactions by actin co-sedimentation assay. The actin co-sedimentation assay is a well-established technique and has been commonly used to demonstrate binding of proteins that interact directly with actin filaments (Ahrens et al., 2012; Mehta and Sibley, 2010; Schuler et al., 2005; Singh et al., 2011). We and others have previously shown that the damaged cell-recognition molecule C-type lectin 9A (Clec9A) recognises a conserved component within nucleated and non-nucleated cells that is exposed only when the cell membrane is damaged (Srivastava and Barber, 2008; Zhang et al., 2012). Here we use Clec9A as an example and present a detailed procedure for demonstrating the direct binding of ...
[摘要] This protocol describes measurement of direct protein-protein interactions by actin co-sedimentation assay. The actin co-sedimentation assay is a well-established technique and has been commonly used to demonstrate binding of proteins that interact directly with actin filaments (Ahrens et al., 2012; Mehta and Sibley, 2010; Schuler et al., 2005; Singh et al., 2011). We and others have previously shown that the damaged cell-recognition molecule C-type lectin 9A (Clec9A) recognises a conserved component within nucleated and non-nucleated cells that is exposed only when the cell membrane is ...
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