title: Expression and purification of bioactive hemagglutinin protein of highly pathogenic avian influenza A (H5N1) in silkworm larvae.
abstract: The hemagglutinin (HA) of avian influenza viruses plays a very important role in the infection of host cells. In this study, the HA gene of the highly pathogenic avian influenza H5N1 virus was cloned and expressed in silkworm larvae. The expressed recombinant HA (rHA) was purified using fetuin-agarose chromatography and Superdex 200 10/300 GL gel filtration chromatography, and the identity of purified rHA was confirmed by SDS-PAGE and Western blot. Approximately 500 μg of purified rHA was obtained from a total of 30 silkworm larvae, suggesting the high efficiency of the silkworm expression system. The purified rHA bound to a rabbit polyclonal antibody against influenza A virus H5N1 (avian flu) HA, suggesting its antigenicity and potential application in vaccine development. Gel filtration chromatography showed that purified HA was present in the void volume fractions, indicating that rHA may form an oligomer. The rHA bound to poly{Neu5Acα2,3LacNAcβ-O[(CH₂)₅NHCO]₂(CH₂)₅NH-/γ-PGA}, which mimics an avian type receptor, but did not bind to γ-polyglutamic acid or human type receptor mimic, poly{Neu5Acα2,6LacNAcβ-O[(CH₂)₅NHCO]₂(CH₂)₅NH-/γ-PGA}, suggesting that it could be utilized as a blocking agent against infection by highly pathogenic influenza viruses.
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Japan.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.forename: Mizuho
authors.initials: M
authors.lastname: Harada
authors.forename: Sawako
authors.initials: S
authors.lastname: Yoshida
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kato
authors.forename: Akiko
authors.initials: A
authors.lastname: Murakawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Ogata
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kato
authors.forename: Taichi
authors.initials: T
authors.lastname: Usui
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics*; D007814:Larva / Q000235:genetics; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011991:Receptors, Virus; D011994:Recombinant Proteins; C109963:hemagglutinin, avian influenza A virus
keywords: Bacmid; Bombyx mori nucleopolyhedrovirus; Hemagglutinin; Influenza virus; Silkworm