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Published on: October 16, 2013
In vitro and in vivo bioactivity of single-chain interleukin-12
D L Foss1, M D Moody, K P Murphy
1University of Minnesota, Department of Veterinary PathoBiology, St. Paul, MN 55108, USA.
Insights
Researchers created a single-chain porcine interleukin-12 (IL-12) protein to simplify studying its immune functions. This bioactive IL-12 enhanced immune responses in both lab tests and animal models, aiding future research.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Interleukin-12 (IL-12) is a cytokine with significant immunoregulatory properties.
- Its heterodimeric structure complicates the study of its function.
- IL-12 is a potential vaccine adjuvant and immune response modulator.
Purpose of the Study:
- To construct a single-chain porcine IL-12 gene for easier study.
- To express recombinant single-chain porcine IL-12 in Pichia pastoris.
- To evaluate the in vitro and in vivo bioactivity of the engineered IL-12.
Main Methods:
- Gene construction and recombinant protein expression in Pichia pastoris.
- In vitro bioactivity assays measuring lymphoblast proliferation and interferon-gamma secretion.
- In vivo oral immunization model to assess antigen-specific IgA and IgG in jejunal mucus.
Main Results:
- Single-chain porcine IL-12 demonstrated bioactivity in vitro on human and porcine cells.
- The p40 subunit alone did not induce proliferation or inhibit single-chain IL-12 activity.
- In vivo, single-chain porcine IL-12 enhanced antigen-specific IgA and IgG in jejunal mucus.
Conclusions:
- Both p40 and p35 subunits are required for IL-12 receptor binding and signal transduction.
- The bioactivity of single-chain IL-12 facilitates its in vivo delivery and study.
- This engineered IL-12 aids in understanding its structure and function in immune modulation.
Abstract:
Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.
