title: Optimization of canine interleukin-12 production using a baculovirus insect cell expression system.
abstract: BACKGROUND
  Interleukin-12 is an important cytokine in mediating cellular immune responses.
  
  
  RESULTS
  Recombinant single-chain canine IL-12 was produced in a baculovirus-insect cell system with the aim of conducting further studies on modulation of immune responses in dogs. To optimize the production of recombinant canine IL-12, a classical baculovirus and a modified vector (chitinase A and v-cathepsin knockout) were used containing a native or an optimized insert of canine IL-12. The optimized IL-12 construct contained the GP64 signal peptide and was synthesized with optimized codons for expression in Trichoplusia ni cells. Dot-blot and Western blot analysis showed the highest production levels of recombinant IL-12 protein by the use of the modified baculovirus vector containing the optimized insert, at a multiplicity of infection of five and at 48 h after infection. The recombinant cytokine was successfully purified and showed a good degree of purity, integrity, folding, and yield, with very little endotoxin contamination. Recombinant canine IL-12 induced IFN-γ in canine lymphocytes, indicating that it was biologically active.
  
  
  CONCLUSIONS
  Therefore, this study describes an efficient method to produce adequate amounts of biologically active canine IL-12, useful for immunomodulation studies in dogs.
authors.affiliation: Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil. cpinheiro@posdoc.bahia.fiocruz.br.|Programa Nacional de Pós Doutorado-CAPES/Programa de Pós-graduação em Biotecnologia em Saúde e Medicina Investigativa, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil. cpinheiro@posdoc.bahia.fiocruz.br.
authors.forename: Cristiane Garboggini Melo
authors.initials: CG
authors.lastname: de Pinheiro
authors.affiliation: Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil. mayaragoes@hotmail.com.
authors.forename: Mayara de Oliveira
authors.initials: Mde O
authors.lastname: Pedrosa
authors.affiliation: Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil. naiara.teixeira@aluno.bahia.fiocruz.br.
authors.forename: Naiara Carvalho
authors.initials: NC
authors.lastname: Teixeira
authors.affiliation: Laboratório de Macromoléculas, Bio-Manguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. Adinis@bio.fiocruz.br.
authors.forename: Ana Paula Dinis
authors.initials: AP
authors.lastname: Ano Bom
authors.affiliation: Laboratory of Virology, Wageningen University, Wageningen, The Netherlands. monique.vanoers@wur.nl.
authors.forename: Monique M
authors.initials: MM
authors.lastname: van Oers
authors.affiliation: Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil. ggileno@bahia.fiocruz.br.|Instituto Nacional de Ciência e Tecnologia de Doenças Tropicais (INCT-DT), Salvador, Brazil. ggileno@bahia.fiocruz.br.
authors.forename: Geraldo Gileno de Sá
authors.initials: GG
authors.lastname: Oliveira
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002688:Chitinases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004285:Dogs; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009036:Moths; D061251:Primary Cell Culture; D017510:Protein Folding; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D018664:Interleukin-12; D002688:Chitinases; D002403:Cathepsins