title: Cloning and expression of a truncated form of envelope glycoprotein D of Bovine herpesvirus type 5 in methylotrophic yeast Pichia pastoris.
abstract: Meningoencephalitis caused by Bovine herpesvirus type 5 (BoHV-5) is responsible for heavy economic losses in the cattle industry. As in other Alphaherpesviruses, the envelope glycoprotein IV (gD), which mediates penetration into host cells, is one of the major candidate antigens for a recombinant vaccine, since it induces a strong and persistent immune response. The DNA coding for a truncated form of BoHV-5 gD (tgD) has been cloned into the Pichia pastoris expression vector pPICZalphaB to allow protein secretion into the medium. After induction with methanol, a approximately 55kDa protein was obtained. Enzyme deglycosylation with Endo H showed a smaller size band in SDS-PGAE, with approximately 50kDa, suggesting that tgD has N-linked oligosaccharides and that it is not hyperglycosylated. The approximately 55kDa protein was recognized by several polyclonal antibodies, including polyclonal antibody anti-tgD and polyclonal antibodies of different animal species immunized with BoHV-5 and BoHV-1. This is the first report of BoHV-5 gD expression in yeast. It was shown that the recombinant truncated form of BoHV-5 gD has antigenic and immunogenic properties similar to the native BoHV-5 gD. Expression of tgD as a secreted protein allows simple and inexpensive purification methods that can be used for further studies to evaluate its immunogenicity in cattle.
authors.affiliation: Centro de Biotecnologia, Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil.
authors.forename: Luana Alves
authors.initials: LA
authors.lastname: Dummer
authors.forename: Fabricio Rochedo
authors.initials: FR
authors.lastname: Conceição
authors.forename: Leandro Quintana
authors.initials: LQ
authors.lastname: Nizoli
authors.forename: Carina Martins
authors.initials: CM
authors.lastname: de Moraes
authors.forename: Andréa Ramos
authors.initials: AR
authors.lastname: Rocha
authors.forename: Lorena Leonardo
authors.initials: LL
authors.lastname: de Souza
authors.forename: Talita
authors.initials: T
authors.lastname: Roos
authors.forename: Telmo
authors.initials: T
authors.lastname: Vidor
authors.forename: Fábio Pereira Leivas
authors.initials: FP
authors.lastname: Leite
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006031:Glycosylation; D025881:Herpesvirus 5, Bovine / Q000235:genetics*; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C091563:glycoprotein D, bovine herpesvirus 5