Development of virus removal technology using virus-binding proteins

致病病毒对水环境的污染引起了人们对病毒感染性疾病在我们社会中爆发的担忧。由于传统的水和废水处理系统不足以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。本项目成功地回收了活性污泥细菌培养物中的病毒结合蛋白(VBP)。将从细菌培养中提取的粗蛋白应用于亲和层析柱，在亲和层析柱中固定来自脊髓灰质炎病毒1型(PV1)的定制衣壳蛋白多肽作为配基，从而实现VBP的回收。酶联免疫吸附试验表明，VBP具有吸附PV1感染性颗粒的能力。VBPs在亲和层析柱上对病毒多肽的吸附具有很强的吸引力，能够克服静电排斥力。双向电泳法显示，分离的VBP含有大量蛋白质，其分子量分布广泛，但均小于100 kDa。对NCBI数据库中所有蛋白质序列的N末端进行同源性搜索，结果表明分离的VBP为新发现的蛋白质。分离了活性污泥菌的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菌DNA文库中分离出VBP基因。证实携带分离到的VBP基因的pRSET转化的大肠杆菌BL21可广泛产生VBP克隆。酶联免疫吸附试验显示，VBP克隆具有与完整的PV1颗粒结合的能力。本研究建立的VBP克隆系统将使大量生产VBP成为可能，并将其用作病毒吸附剂的新材料。

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.