Development of virus removal technology using virus-binding proteins

致病性病毒对水环境的污染引起了人们对我们社会中病毒性传染病爆发的关注。由于传统的水和废水处理系统不足以有效地灭活或去除致病病毒，因此需要开发一种新的病毒去除技术。在本项目中，从活性污泥中提取的细菌培养物中成功地回收了病毒结合蛋白（VBPs）。通过将细菌培养物中提取的粗蛋白应用于亲和柱上，将定制的1型脊髓灰质炎病毒（PV1）衣壳蛋白肽固定为配体，实现了vbp的回收。ELISA检测表明，vps具有吸附PV1感染颗粒的能力。在亲和柱中，vbp对病毒肽的吸附具有强大的吸引力，能够克服静电斥力。双向电泳结果表明，分离得到的VBPs含有多种蛋白质，分子量分布广泛，但均小于100kDa。NCBI数据库中所有蛋白序列的N端同源性分析表明，分离得到的vbp均为新发现的蛋白。分离了活性污泥细菌的VBP基因，建立了VBP的克隆体系。利用菌落杂交技术从活性污泥细菌的DNA文库中分离出VBP基因。结果表明，携带分离的VBP基因的pRSET转化大肠杆菌BL21可大量产生VBP克隆。酶联免疫吸附试验显示，VBP克隆具有与PV1完整颗粒结合的能力。本研究建立的VBP克隆系统将使VBP的大批量生产成为可能，并使其成为一种新型的病毒吸附剂材料。

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.