Cloning and expression of a truncated form of envelope glycoprotein D of Bovine herpesvirus type 5 in methylotrophic yeast Pichia pastoris.
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.
Dummer
Luana Alves
LA
Centro de Biotecnologia, Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil.
Conceição
Fabricio Rochedo
FR
Nizoli
Leandro Quintana
LQ
de Moraes
Carina Martins
CM
Rocha
Andréa Ramos
AR
de Souza
Lorena Leonardo
LL
Roos
Talita
T
Vidor
Telmo
T
Leite
Fábio Pereira Leivas
FP
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
D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C091563:glycoprotein D, bovine herpesvirus 5