Development of virus removal technology using virus-binding proteins

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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.