title: Expression and characterization of bluetongue virus serotype 21 VP7 antigen: C-terminal truncated protein has significantly reduced antigenicity.
abstract: In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.
authors.affiliation: National Institute of Animal Health, 3–1–5 Kan-non-dai, Tsukuba, Ibaraki 305–0856, Japan.
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Hosamani
authors.forename: Shinya
authors.initials: S
authors.lastname: Shimizu
authors.forename: Jiro
authors.initials: J
authors.lastname: Hirota
authors.forename: Takehiro
authors.initials: T
authors.lastname: Kokuho
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kubota
authors.forename: Satoko
authors.initials: S
authors.lastname: Watanabe
authors.forename: Masato
authors.initials: M
authors.lastname: Ohta
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Muneta
authors.forename: Shigeki
authors.initials: S
authors.lastname: Inumaru
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism; D001819:Bluetongue / Q000453:epidemiology / Q000821:virology*; D001820:Bluetongue virus / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D002418:Cattle Diseases / Q000453:epidemiology / Q000821:virology*; D002460:Cell Line; D006224:Cricetinae; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D007564:Japan / Q000453:epidemiology; D012703:Serotyping; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000956:Antigens, Viral; D014758:Viral Core Proteins; C066862:VP7 protein, orbivirus