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Analysis of the antigen epitopes of infectious pathogens in laboratory animals using peptide tips

使用肽尖分析实验动物感染性病原体的抗原表位

基本信息

批准号：
15300138
负责人：
AGUI Takashi
金额：
$ 10.69万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15300138/
关键词：
peptide tip antigenic epitope hantavirus mouse hepatitis virus Sendai virus Mycoplasma マイコフラズマ

项目摘要

We selected mouse hepatitis virus, Sendai virus, Mycoplasma plumonis, and hantavirus from the aspect of the frequency and danger upon infectious accidents. We determined antigenic epitopes by incubating polyclonal anti-sera with peptide tips produced according to the amino acid sequences reported previously elsewhere. Further, we tried to develop a preliminary ELISA kit using antigenic epitopes that we have determined. With respect to mouse hepatitis virus and Sendai virus, we succeeded to develop an ELISA system, which was more sensitive or equal to the commercially available ELISA system (paper in preparation). With respect to hantavirus, we succeeded to determine an antigenic epitope, EDVNGIRK (amino acid 166-173), to monoclonal antibody E5/G6, which recognizes nuclear protein of hantavirus. With respect to Mycoplasma plumonis, we first searched antigenic protein from the components of the bacteria, since there are a lot of proteins composing of bacteria. We found a protein showing a homology to P46 protein, which had been reported as an antigenic protein in other Mycoplasmas and named it P46L. We produced recombinant P46L protein conjugated with glutathione S-transferase and made an ELISA system with purified P46L. This ELISA system was more sensitive than the commercially available ELISA system.We further applied this method to the determination of antigenic epitopes of West Nile virus and SARS virus and succeeded it. We furthermore succeeded to apply this method to not only determining antigenic epitopes of infectious pathogens but also determining protein-protein interaction site such as binding sits of FAS-antigen and angiotensin II.

从发生频率和对传染性事故的危险性方面，我们选择了小鼠肝炎病毒、仙台病毒、羽支原体和汉坦病毒。我们确定的抗原表位，通过孵育多克隆抗血清与肽尖根据先前在别处报道的氨基酸序列产生。此外，我们尝试使用我们已经确定的抗原表位开发初步的ELISA试剂盒。对于小鼠肝炎病毒和仙台病毒，我们成功开发了一种ELISA系统，该系统比市售ELISA系统（论文正在编写中）更敏感或相当。对于汉坦病毒，我们成功地确定了单克隆抗体E5/G6的抗原表位EDVNGIRK（氨基酸166-173），其识别汉坦病毒的核蛋白。对于羽支原体，由于细菌的蛋白质组成很多，我们首先从细菌的组分中寻找抗原蛋白。我们发现了一个与P46蛋白具有同源性的蛋白，该蛋白已被报道为其他支原体的抗原蛋白，并将其命名为P46 L。我们制备了谷胱甘肽S-转移酶标记的重组P46 L蛋白，并与纯化的P46 L建立了ELISA体系。该方法不仅用于确定感染性病原体的抗原表位，而且还可用于确定FAS-抗原与血管紧张素II的结合位点等蛋白质-蛋白质相互作用位点。