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Studies on the development of diagnosis for infectious diseases among laboratory rodents

实验啮齿类动物传染病诊断研究进展

基本信息

批准号：
11558096
负责人：
ARIKAWA Jiro
金额：
$ 8万
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：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11558096/
关键词：
hemorrhagic fever with renal syndrome HFRS MHV LCM diagnosis laboratory animal zoonosis antibody 賢症候性出血熱

项目摘要

1. Nucleocapsid (N) proteins of Hantaan, Seoul and Dobrava viruses were expressed by E. coli and baculovirus systems. The recombinant proteins by both systems produced enough amount of recombinant proteins and retained the anti genicity similar to those of authentic viruses. They are considered to be a ble to apply for ELISA antigen.2. The truncated N proteins which lacked 50 amino acids at N-terminal reduced the cross reactivity to antibody to heterologous serotypes. Therefore, the truncated antigens were applied as serotyping antigen.The ELISA with the truncated antigen able to serotype of patient and rodent sera from China and Far Eastern part of Russia as determined by ordinary neutralization test.3. Mouse hepatitis virus N protein gene was expressed by yeast system of which maximum expression was observed at 72 hours after culture. However, the antigenicity was too low to apply for ELISA antigen.4. cDNA of N protein of lymphocytic choriomeningitis (LCM) virus were cloned from Japanese isolate (strain OQ28) and prototype strain (strain WE). The cDNAs of full length, C-terminal region and central region were independently transfected to COS cells. Although they could express recombinant proteins, amount of antigen expressed was too small to apply to ELISA.5. Recombinant baculovirus expressing LCM virus N protein was provided from National Institute of Infectious Diseases Japan. Both the recombinant baculovirus infected SF-9 cells and Tm5 cells were found to be applicable for IFA antigen and ELISA antigen, respectively. A total of 9,840 mouse sera from 1,117 animal facility in Japan were tested for LCM virus antibody by IFA and ELISA. From these results, screening with ELISA followed by confirmation by IFA was recommended.

1.利用大肠杆菌和杆状病毒系统表达了汉滩病毒、首尔病毒和多布拉瓦病毒的核衣壳蛋白。两种系统得到的重组蛋白都能产生足够数量的重组蛋白，并保持了与正品病毒相似的抗原性。结论：1.它们被认为是一种很好的申请ELISA抗原的方法。N端缺失50个氨基酸的截短N蛋白降低了与异种血清型抗体的交叉反应性。因此，将截短抗原作为血清学分型抗原，用常规中和试验对中国和远东地区的患者和鼠类血清进行分型。小鼠肝炎病毒N蛋白基因在酵母系统中得到了表达，培养72小时达到最高表达。但其抗原性较低，不能用于ELISA抗原性检测。从日本分离株(OQ28株)和原型株(WE株)中克隆了淋巴细胞性脉络膜脑膜炎病毒(LCM)N蛋白基因。将全长、C端区和中心区的cDNA分别导入COS细胞。虽然它们可以表达重组蛋白，但表达的抗原量太少，不适用于ELISA。表达LCM病毒N蛋白的重组杆状病毒由日本国立传染病研究所提供。重组杆状病毒感染的SF-9细胞和TM5细胞分别适用于IFA抗原和EL ISA抗原。用间接免疫荧光法和酶联免疫吸附试验检测了来自日本1117家动物机构的9840份小鼠血清中的LCM病毒抗体。根据这些结果，建议先用ELISA法进行筛查，然后用IFA进行确认。