Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle.
Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil. Electronic address: bssiedler@gmail.com.
Bianca S
BS
Siedler
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Bárbara C
BC
Roloff
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Gizele L
GL
de Sá
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Alessandra
A
Neis
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Fabrício R
FR
Conceição
Instituto de Biologia, Departamento de Microbiologia e Parasitologia, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Daiane D
DD
Hartwig
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Sibele
S
Borsuk
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Odir A
OA
Dellagostin
Instituto de Ciências Básicas e da Saúde, Universidade Federal do Rio Grande do Sul, Av. Sarmento Leite 500, 90050-170, Porto Alegre, RS, Brazil.
Fabrício S
FS
Campos
Instituto de Ciências Básicas e da Saúde, Universidade Federal do Rio Grande do Sul, Av. Sarmento Leite 500, 90050-170, Porto Alegre, RS, Brazil.
Paulo M
PM
Roehe
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil.
Claudia P
CP
Hartleben
Núcleo de Biotecnologia - CDTec, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, 96010-900, Pelotas, RS, Brazil; Centro de Pesquisa Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, BA, Brazil.
Alan J A
AJ
McBride
D000818:Animals; D002417:Cattle; D015870:Gene Expression; D007242:Herpesvirus 1, Bovine / Q000235:genetics* / Q000378:metabolism; D025881:Herpesvirus 5, Bovine; D022283:Herpesvirus Vaccines / Q000494:pharmacology; D007241:Infectious Bovine Rhinotracheitis / Q000097:blood / Q000175:diagnosis; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D022283:Herpesvirus Vaccines; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins; C414993:glycoprotein E, bovine herpesvirus type 5
Bovine herpetic encephalitis; Differential vaccine; Immunoassay; Infectious bovine rhinotracheitis; Infectious pustular balanoposthitis; Infectious pustular vulvovaginitis