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

选取小鼠肝炎病毒、仙台病毒、毛囊支原体和汉坦病毒，从感染事故发生的频率和危险性方面进行分析。我们通过培养具有肽尖的多克隆抗血清来确定抗原表位，这些肽尖是根据先前在其他地方报道的氨基酸序列产生的。此外，我们尝试使用我们已经确定的抗原表位开发一种初步的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灵敏度更高或与市售ELISA系统相当的ELISA系统（论文正在准备中）。对于汉坦病毒，我们成功地确定了识别汉坦病毒核蛋白的单克隆抗体E5/G6的抗原表位EDVNGIRK（氨基酸166-173）。对于plumonis支原体，我们首先从细菌的组成成分中寻找抗原蛋白，因为组成细菌的蛋白质很多。我们发现了一个与P46蛋白同源的蛋白，该蛋白在其他支原体中已报道为抗原蛋白，并命名为P46L。我们制备了谷胱甘肽s -转移酶结合的重组P46L蛋白，并用纯化的P46L构建了ELISA体系。该酶联免疫吸附测定系统的灵敏度高于市售酶联免疫吸附测定系统。我们进一步将该方法应用于西尼罗病毒和SARS病毒抗原表位的测定，并取得了成功。此外，我们还成功地将该方法应用于感染性病原体抗原表位的测定，以及fas抗原和血管紧张素II结合位点等蛋白-蛋白相互作用位点的测定。

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.