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Establishment of electron microscopic in situ hybridization for viral nucleic acids

病毒核酸电镜原位杂交技术的建立

基本信息

批准号：
09670240
负责人：
GOTO Toshiyuki
金额：
$ 1.86万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670240/
关键词：
in situ hybridization electron microscopy contract enhancement viral nucleic acids Epstein-Barr virus human immunodeficiency virus(HIV)Borna disease virus adenovirus in situハイブリダイゼーションコントラスト増強 PCR

项目摘要

The nucleic acids and proteins of new viruses have been elucidated by recent advance of molecular biology, but their structures are still unclear. The morphological structure is very important to know the basic natures of the viruses. To elucidate the structure of the newly found viruses, we have been developing an electron microscopic in situ hybridization (EM-ISH) technique for identifying viral nucleic acids in the following items these years.1.To establish EM-ISH, we observed Epstein-Barr virus (EBV)-infected cells by using EBV BamHl-W as a probe. The conditions and detection system for EM-ISH in EBV were established in lowtemperature embedded sections, and found to be viral precursors in the cells.2.We obtained high contrast images by staining with ruthenium red and OsO_4 solution afterimmunolabeling on the low-temperature embedded sections.3.The nucleic acids in the core of HIV particles were found by EM-ISH.4.The paracrystalline arrays of adenovirus in the infected cells were found to be a standard sample todevelop the fundamental conditions and detection systems for EM-ISH.5.One labeling per one or a fexv copies of adenovirus DNA can be detected by EM-ISH with using PCR to make highly biotin-labeled probes. The efficient labeling for HIV has been improved on the way.6.The structure and morphogenesis of Borna disease virus (BDV) have been found by immunoelectron microscopy. Further studies on BDV are being carried out by EM-ISH.

近年来，分子生物学的进展已阐明了新病毒的核酸和蛋白质，但其结构仍不清楚。病毒的形态结构对了解病毒的基本性质具有重要意义。为了阐明新发现的病毒的结构，近年来，我们一直在发展用于鉴定病毒核酸的电子显微镜原位杂交（EM-ISH）技术，具体如下：1.为了建立EM-ISH，我们用EB病毒（EBV）BamHl-W作为探针观察了EBV感染的细胞。建立了EB病毒低温包埋切片EM-ISH的条件和检测体系，2.低温包埋切片免疫标记后钌红和OsO_4溶液染色，获得高对比度的图像; 3.电镜观察发现HIV颗粒核心有核酸存在; 4.电镜观察发现HIV颗粒核心有病毒前体存在; 5.电镜观察发现HIV颗粒核心有病毒前体存在。4.感染细胞中腺病毒的类晶体阵列可作为建立EM-ISH的基本条件和检测系统的标准样品。5.每一个或一个fexv拷贝的腺病毒DNA可被EM-ISH检测到一个标记。原位杂交结合PCR制备高生物素标记探针。6.利用免疫电镜技术研究了博尔纳病病毒（Borna disease virus，BDV）的结构和形态发生。EM-ISH正在对BDV进行进一步研究。