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Optimization of canine interleukin-12 production using a baculovirus insect cell expression system
Cristiane Garboggini Melo de Pinheiro1,2, Mayara de Oliveira Pedrosa3, Naiara Carvalho Teixeira4
1Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil. cpinheiro@posdoc.bahia.fiocruz.br.
BMC Research Notes
|January 23, 2016
Summary
Researchers efficiently produced biologically active recombinant canine interleukin-12 (IL-12) using an optimized baculovirus vector. This method yields sufficient quantities for canine immunomodulation studies.
Area of Science:
- Veterinary Immunology
- Molecular Biology
- Biotechnology
Background:
- Interleukin-12 (IL-12) is a critical cytokine for cellular immune responses.
- Understanding and manipulating canine immune responses requires specific tools.
Purpose of the Study:
- To develop an efficient method for producing recombinant single-chain canine IL-12.
- To optimize the production of canine IL-12 using baculovirus expression systems.
Main Methods:
- Utilized classical and modified baculovirus vectors with native or optimized canine IL-12 inserts.
- Optimized expression in Trichoplusia ni cells using GP64 signal peptide and codon optimization.
- Analyzed protein production levels via dot-blot and Western blot.
Main Results:
- A modified baculovirus vector with an optimized insert yielded the highest recombinant canine IL-12 production.
- Optimal production achieved at a multiplicity of infection of five and 48 hours post-infection.
- Purified recombinant canine IL-12 was biologically active, inducing IFN-γ in canine lymphocytes.
Conclusions:
- An efficient method for producing adequate amounts of biologically active canine IL-12 was established.
- The produced canine IL-12 is suitable for immunomodulation studies in dogs.
