Construction of a Cloning System for the Mass Production of a Virus-Binding Protein Specific for Poliovirus Type 1

Abstract

In our previous study, virus-binding proteins (VBPs) demonstrating the ability to strongly bind poliovirus type 1 (PV1) were recovered from a bacterial culture derived from activated sludge. The isolated VBPs would be useful as viral adsorbents for water and wastewater treatments. 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. The sequence of the VBP gene consisted of 807 nucleotides encoding 268 amino acids. Fifteen amino acid sequences were retrieved from 2,137,877 sequences by a homology search using the BLAST server at the National Center for Biotechnology Information. The protein encoded in the isolated genome was considered to be a newly discovered protein from activated sludge culture, because any sequences in protein databases were not perfectly matched with the sequence of the VBP. It was confirmed that Escherichia coli BL21 transformed by pRSET carrying the isolated VBP gene could extensively produce the VBP clones. Enzyme-linked immunosorbent assay (ELISA) revealed that the VBP clone exhibited the binding ability with intact particles of PV1. The equilibrium binding constant between PV1 and VBP in the ELISA well was estimated to be 2.1 ؋ 10 7 (M ؊1 ), which also indicated that the VBP clones have a high affinity with the PV1 particle. The VBP cloning system developed in this study would make it possible to produce a mass volume of VBPs and to utilize them as a new material of the specific adsorbent in several technologies, including virus removal, concentration, and detection.Waterborne infectious diseases caused by pathogenic viruses such as enteroviruses, adenoviruses, rotaviruses, and noroviruses have been frequently reported even in the developed countries, where hygiene conditions have been considerably improved (5-7, 12, 21, 26, 27). These pathogenic viruses can survive for a long period in water environments and transmit to humans (7, 10), so a public health hazard is posed by the spread of pathogenic viruses in water environments. Furthermore, the subsistence of these viruses in our society would raise concerns about viruses with virulent phenotypes by mutation or recombination, as has been reported with respect to vaccine-derived polioviruses (8,15,20) and the other several RNA viruses (22)(23)(24)38). In order to prevent pathogenic viral contamination of the water environment, it would be significant to remove or inactivate pathogenic viruses in water and wastewater treatment processes. However, the conventional water and wastewater treatment systems have difficulty in removing or inactivating viruses. It is well known that the filtration process, the most fundamental technology in the water treatment system, is not operative in removing viruses (18). Several viruses have a higher resistance to disinfectants than indicator microorganisms (28). A new technology for virus removal needs to be developed in...

Adsorption characteristics of an enteric virus-binding protein to norovirus, rotavirus and poliovirus

“…A genomic DNA of activated sludge microorganisms was extracted as previously described [ 15 ]. Extracted genomic DNA of activated sludge microorganisms was treated with DNase-free RNase A and purified with phenol/chloroform and 100% ethanol.…”

“…Purified EVBP candidate was desalted by dialysis against 20 mM NH 4 HCO 3 (pH: 8.0) at 4°C for at least 12 hr, and concentrated with a vacuum and centrifugal dehydrator (CVE-100, EYELA, TOKYO RIKAKIKAI CO. Ltd., Tokyo, Japan). EVBP candidate in the pellet was suspended in 100 μl of double-autoclaved milliQ water, and processed for SDS-PAGE and silver staining as described previously [ 15 ].…”

“…Virus-binding proteins, which were discovered from a bacterial culture derived from activated sludge [ 14 ], could be alternative viral adsorbents, because these bacterial proteins can stably interact with human viral particles [ 15 ]. Recently, norovirus-binding proteins (NoVBPs) were acquired with an affinity to a norovirus (NoV) capsid peptide [ 16 ].…”