Development of new technology for removing viruses from water environment

病毒结合蛋白(VBP)是人类病毒在水环境中优先吸附的细菌蛋白。以病毒衣壳多肽为配基，用亲和层析法从活性污泥细菌培养物中发现VBP。本研究的目的是开发一种利用VBPS从环境水中去除病毒的新技术。在为期3年的研究中取得了以下成果。在研究的第一年，为了获得大量的VBP，进行了VBP的生产和纯化的放大实验。构建了一种手工纯化VBP的色谱柱，可获得比常规方法大约10倍的VBP。第二年，利用聚合酶链式反应构建了C端有6个连续赖氨酸残基的十六氨酸标记VBP基因(LysTagVBP)，并用于在大肠杆菌中表达LysTagVBP。然后用硅烷偶联剂和戊二醛将LysTagVBP中的聚赖氨酸区偶联到玻璃颗粒表面。LysTagVBP的生成直接关系到VBP在载体表面的有效固定化，在研究的第三年，构建了固定化LysTagVBP的脱毒柱，并对其脱毒效果进行了评价。结果，在最初的1小时内，病毒减少了约40%，3小时后，70%的病毒被清除。VBPS除病毒技术的发展是一个全新的挑战，VBPS作为病毒吸附剂的研究成果将有助于VBPS在水中病毒去除方面的进一步应用。

Virus-binding proteins (VBPs) are bacterial proteins to which human viruses preferentially adsorb in water environment. VBPs were discovered from a bacterial culture derived from activated sludge with affinity chromatography assay in which a viral capsid peptide was used as a ligand. The purpose of this study was to develop a novel technology using VBPs for virus removal from environmental water. Achievements described below were obtained during the 3-year study duration.At the first year of the study duration, the scale-up of VBP production and purification was conducted in order to obtain large amount of VBP. A handmade column for VBP purification was constructed, which made it possible to obtain about 10 times larger volume of VBP than conventional procedure.At the second year, gene of hexalysine-tagged VBP (LysTagVBP), which has 6-consecutive lysine residues in its C terminus, was fabricated with PCR, and used for production of LysTagVBP with E. coli. Then, polylysine region in LysTagVBP was coupled with the surface of glass particles using silane coupling reagent and glutaraldehyde. The creation of LysTagVBP directly linked to the effective immobilization of VBP on carrier surface.At the third year of the study duration, the virus-removing column in which LysTagVBP was immobilized was constructed, and virus removal efficiency with the column was evaluated. As a result, about 40% of viruses were reduced during the first 1-hour operation, and 70% of viruses were removed after the 3-hour operation. The development of virus removal technology using VBPs is a totally new challenge, and the achievements with regards to the utilization of VBPs as viral adsorbent would be useful in the farther application of VBPs to virus removal from water.