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Published on: December 28, 2015
Detection, cDNA cloning and sequencing of canine interleukin 12
M Büttner1, G Belke-Louis, H J Rziha
1Institute of Vaccines, Federal Research Centre for Virus Diseases of Animals, Tuebingen, Germany. Buettner@tue.bfav.de
Insights
Researchers identified interleukin 12 (IL-12) in dogs using RT-PCR and sequencing. Canine IL-12 shows high similarity to other mammals, providing a basis for future canine immunology research.
Area of Science:
- Immunology
- Molecular Biology
- Canine Genetics
Background:
- Interleukin 12 (IL-12) is a key cytokine regulating immune responses.
- Understanding canine IL-12 is crucial for advancing veterinary immunology and therapeutics.
Purpose of the Study:
- To identify and characterize the mRNA and nucleotide sequences of canine interleukin 12 (IL-12) subunits.
- To investigate the induction of canine IL-12 mRNA synthesis by microbial stimuli.
- To compare the canine IL-12 sequence with other mammalian species.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) for mRNA identification.
- Rapid amplification of cDNA ends (RACE)-PCR and cDNA cloning for full sequence determination.
- Bioinformatic analysis for amino acid sequence comparison.
Main Results:
- Canine IL-12 mRNA was detected in peripheral blood mononuclear cells (PBMC).
- Stimulation with Staphylococcus aureus and inactivated vaccinia virus induced IL-12 mRNA synthesis.
- Canine IL-12 subunits exhibited over 80% amino acid identity with other mammals, particularly humans.
- Canine IL-12 did not cross-react with anti-human IL-12 antibodies.
- Canine PBMC supernatants induced interferon gamma (IFN-gamma) mRNA, with recombinant human IL-12 also being an inducer.
Conclusions:
- The cloning and sequencing of canine IL-12 provide foundational data for further research.
- Canine IL-12 plays a role in immune regulation, similar to its counterparts in other species.
- Future studies can explore the biological functions and therapeutic potential of canine IL-12.
Abstract:
In canine peripheral blood mononuclear cells (PBMC) the mRNAs coding for both subunits of canine interleukin 12 (IL-12) were identified using reverse transcription polymerase chain reaction (RT-PCR). Stimulation of canine PBMC with Staphylococcus aureus strain Cowan plus Concanavalin A for 5 h resulted in significant mRNA synthesis. Likewise, inactivated vaccinia virus induced IL-12 mRNA synthesis, however with different kinetics. The complete nucleotide sequence for both IL-12 subunits was determined using rapid amplification of cDNA ends (RACE)-PCR and cloning of amplified specific cDNAs. Computer-aided amino acid (aa) sequence comparison of both canine IL-12 subunits revealed more than 80% identity with the amino acid sequences of six other mammalian species. Closest relationship was found to human, porcine, bovine and cervine IL-12. However, no reactivity was found with antibodies directed against human IL-12, when supernatants of stimulated canine PBMC were tested. Supernatants of canine PBMC stimulated for IL-12 release also induced interferon gamma (IFN-gamma) mRNA as detectable by RT-PCR; however, it was not clear whether IFN-gamma mRNA synthesis was due to an IL-12 specific effect or other stimuli. As to the stimulating effect of IL-12 on canine IFN-gamma mRNA synthesis, recombinant human IL-12 was found to be a good inducer. Since IL-12 is regarded a major regulatory molecule of T-cell-mediated immune response and cell growth our work on the cloning and sequencing of this cytokine from dogs lays the basis for future investigations on the biological and possible therapeutic role of canine IL-12.
