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Published on: January 15, 2011
Cloning and expression of feline interleukin 15
Gregg A Dean1, Anne Barger, Alora Lavoy
1Department of Molecular Biomedical Sciences, 4700 Hillsborough Street, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA. gregg_dean@ncsu.edu
Insights
Researchers successfully cloned and expressed biologically active feline interleukin 15 (IL15), crucial for feline immune studies. This advancement enables further research into feline infectious diseases and immune responses.
Area of Science:
- Veterinary Immunology
- Molecular Biology
- Virology
Background:
- Feline infectious peritonitis virus (FIPV) infection impacts feline health.
- Interleukin 15 (IL15) is vital for immune cell function.
- Understanding feline IL15 is crucial for FIPV research.
Purpose of the Study:
- To clone and characterize the feline interleukin 15 (IL15) cDNA.
- To express biologically active recombinant feline IL15.
- To facilitate studies on feline immune responses.
Main Methods:
- Cloning of feline IL15 cDNA from infected cat lymph nodes.
- Expression of recombinant feline IL15 in Escherichia coli and HEK293T cells.
- Functional assays using feline lymphocytes and CTLL-2 cells; antibody cross-reactivity studies.
Main Results:
- Feline IL15 cDNA (486 bp) encodes a 162-amino acid protein.
- Optimized expression in HEK293T cells yielded biologically active feline IL15.
- Recombinant feline IL15 sustained feline lymphocytes and induced CTLL-2 cell proliferation.
- Antibodies against human IL15 recognized feline IL15; ELISA detection was possible.
Conclusions:
- Biologically active feline IL15 was successfully expressed and characterized.
- The recombinant protein is functional and cross-reactive with human IL15 antibodies.
- This provides a valuable tool for feline immunology and FIPV research.
Abstract:
A cDNA encoding feline interleukin 15 (IL15) was cloned from the lymph node of a cat infected with feline infectious peritonitis virus. The cDNA is 486 bp in length and encodes a protein of 162 amino acids. Recombinant protein was readily expressed as a GST fusion in Escherichia coli and purified by glutathione affinity chromatography. Expression of recombinant protein in mammalian cells was only accomplished by eliminating the 5' and 3' UTR, replacing the IL15 signal peptide with the tissue plasminogen activator signal peptide, and adding 3' sequence to disrupt presumptive secondary structure of the mRNA. Biologically active feline IL15 was expressed in HEK293T cells and was shown to sustain primary feline lymphocytes, a feline T cell line, and mouse CTLL-2 cells. Proliferation of CTLL-2 cells was induced by the recombinant protein in a dose-dependent manner. Monoclonal and polyclonal antibodies against human IL15 recognized feline IL15 in immunofluorescence and Western blot assays. Additionally, feline IL15 was detectable using a commercially available human IL15 ELISA kit.
