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Development of virus removal technology using virus-binding proteins

开发利用病毒结合蛋白的病毒去除技术

基本信息

批准号：
13555145
负责人：
OMURA Tatsuo
金额：
$ 8.64万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral Infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this project, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from poliovirus type 1 (PV1) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 as determined by ELISA. The adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular weights were widely distributed but smaller than 100kDa. Homology searches for the N termini against all protein sequences in NCBI database showed that the isolated VBPs were newly discovered proteins. The VBP gene of activated sludge bacteria was isolated, and the cloning system of the VBP was established. The isolation of the VBP gene from DNA libraries for activated sludge bacteria was achieved with the colony hybridization technique. It was confirmed that E. coli BL21 transformed by pRSET carrying the isolated VBP gene could extensively producethe VBP clones. ELISA revealed that the VBP clone exhibited the binding ability with intact particles of PV1. The VBP cloning system developed in this study would make it possible to produce a mass volume of the VBP, and to utilize them as a new material of the viral adsorbent.

病原性病毒对水环境的污染引起了人们对病毒性传染病暴发的关注。由于传统的水和废水处理系统不足以有效地抑制或去除致病病毒，因此需要开发一种新的病毒去除技术。在这个项目中，病毒结合蛋白（VBP）的细菌培养物来自活性污泥中成功地回收。通过将从细菌培养物中提取的粗蛋白应用于亲和柱来实现VBPs的回收，在亲和柱中，来自脊髓灰质炎病毒1型（PV1）的衣壳蛋白的定制肽被固定作为配体。通过ELISA测定，VBPs表现出吸附PV1感染性颗粒的能力。在亲和柱中，VBPs与病毒肽的吸附以能够克服静电排斥力的强吸引力发生。双向电泳结果表明，所分离的VBPs含有多种蛋白质，分子量分布较广，但均小于100kDa。与NCBI数据库中的所有蛋白质序列进行同源性搜索，结果表明所分离的VBPs为新发现的蛋白质。分离了活性污泥细菌VBP基因，建立了VBP基因克隆系统。采用殖民地杂交技术从活性污泥细菌DNA文库中分离到VBP基因。证实E. pRSET转化大肠杆菌BL21，可大量获得VBP克隆。ELISA结果显示，VBP克隆具有与完整的PV1颗粒结合的能力。本研究所建立的VBP克隆系统，为大规模生产VBP，并将其作为新型病毒吸附材料提供了可能。