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首页> 外文学位 >Expression and characterization of the human neuronal cannabinoid receptor CB1 using the Semliki Forest virus.
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Expression and characterization of the human neuronal cannabinoid receptor CB1 using the Semliki Forest virus.

机译：使用Semliki Forest病毒表达和表征人神经元大麻素受体CB1。

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摘要

The Semliki Forest Virus gene expression system was used to express the human neural cannabinoid receptor CB1. CB1 cDNA was sub-cloned from pSKCANR into the pSFV1 expression vector creating pSFV1-CB1. RNA transcribed in vitro from pSFV1-CB1 was co-transfected into BHK-21 cells with pSFV-HELPER2 RNA to generate recombinant SFV-CB1 virus particles. BHK-21 cells infected with recombinant virus particles expressed high levels of CB1 as determined by Northern blot analysis, Western immunobloting using antibodies elicited to the CB1, and Scatchard-Rosenthal analysis of [3H]CP 55,940 receptor binding of membrane preparations. In addition, Scatchard-Rosenthal analysis indicated expression of CB 1 binding sites with two different affinities for ligand. The high affinity site indicated a Kd of 0.99 +/- 0.3 nM and a Bmax of 10.9 +/- 3.4 pmol/mg, while the low affinity site indicated a K d of 1.6 +/- 0.3 nM and a Bmax of 18.9 +/- 5.1 pmol/mg consistent with high level expression of receptors. Immunocytochemical studies indicated that CB1 exhibited cytoplasmic punctations with accumulations at distal cellular extensions in a pattern which mimicked that found in primary rat brain cells and tissues. In addition, immunocytochemical analysis indicated a distribution for CB1 that was distinct from that of CB2 expressed in the same system indicative of implied functional differences. Immunohistochemical analysis also demonstrated staining for CB1 along filamentous networks in the cytoplasm suggestive of an association with actin, but not with tubulin or glial fibrillary acidic protein. In addition, the CB1 colocalized with the Golgi marker, gamma-adaptin and the transport of CB1 from the endoplasmic reticulum to the Golgi was associated with brefeldin A-sensitive vesicles. Collectively, these results indicate that the Semliki Forest Virus expression system is a viable means of expressing cannabinoid receptors in a state that closely mimics that of the native receptors.
机译：Semliki森林病毒基因表达系统用于表达人类神经大麻素受体CB1。将CB1 cDNA从pSKCANR亚克隆到pSFV1表达载体中，创建pSFV1-CB1。从pSFV1-CB1体外转录的RNA与pSFV-HELPER2 RNA共转染到BHK-21细胞中，产生重组SFV-CB1病毒颗粒。重组病毒颗粒感染的BHK-21细胞表达高水平的CB1，这是通过Northern印迹分析，使用针对CB1的抗体进行的Western免疫印迹以及膜制剂的[3H] CP 55,940受体结合的Scatchard-Rosenthal分析确定的。另外，Scatchard-Rosenthal分析表明CB 1结合位点的表达对配体具有两种不同的亲和力。高亲和力位点表明Kd为0.99 +/- 0.3 nM，Bmax为10.9 +/- 3.4 pmol / mg，而低亲和力位点表明Kd为1.6 +/- 0.3 nM和Bmax为18.9 + / -5.1 pmol / mg与受体的高水平表达相一致。免疫细胞化学研究表明，CB1表现出胞质点状，在远端细胞延伸处积累，其模式类似于在原代大鼠脑细胞和组织中发现的模式。此外，免疫细胞化学分析表明CB1的分布与在同一系统中表达的CB2的分布不同，表明存在隐含的功能差异。免疫组织化学分析还显示沿细胞质中的丝状网络染色CB1，提示与肌动蛋白有关，但与微管蛋白或神经胶质原纤维酸性蛋白无关。此外，CB1与高尔基体标记，γ-adaptin共定位，并且CB1从内质网向高尔基体的运输与布雷菲德菌素A敏感囊泡有关。总的来说，这些结果表明，Semliki森林病毒表达系统是一种表达大麻素受体的可行方法，其状态与天然受体的状态极为相似。

著录项

  • 作者

    Olson, John Mark.;

  • 作者单位

    Virginia Commonwealth University.;

  • 授予单位 Virginia Commonwealth University.;
  • 学科 Biology Neuroscience.; Biology Cell.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 1999
  • 页码 165 p.
  • 总页数 165
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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