|Smith, C wayne|
Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/6/2005
Publication Date: 8/15/2005
Citation: Xiong, C., Hixson, P.M., Mendoza, L.H., Smith, C.W. 2005. Cloning and expression of rabbit interleukin-15. Veterinary Immunology and Immunopathology. 107:131-141. Interpretive Summary: IL-15 is a chemokine (a class of hormones) that has the ability to alter immune cells and the inflammatory process. It is produced by endothelial cells (the cells that line the blood vessels), and is released into the blood and tissues at the time white blood cells are crawling out of the blood into the tissue. In our efforts to understand possible mechanisms whereby dietary factors such as high fat and high sugar diets cause inflammation, we have been developing the experimental tools to analyze possibly important genes. This paper describes the technical details of how this was accomplished with this specific important chemokine.
Technical Abstract: In order to understand the inflammatory mechanisms related to rabbit interleukin-15 (RIL-15), we cloned and expressed RIL-15 cDNA gene. A cDNA encoding RIL-15 was cloned from heart mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using hIL-15 primers. The RIL-15 cDNA contains an open reading frame (ORF) of 162 amino acids (aa) with a 48 aa leader sequence. The predicted molecular weight of the encoded protein (12.5 kDa) matched the size of recombinant IL-15 on Western blotting in an Escherichia coli (pET32a) expression system. Amino acid and nucleotide sequence analyses of RIL-15 revealed 82.7% and 87% homology with human IL-15 (hIL-15), respectively. RIL-15 is similar to the hIL-15 (hIL-15) in that it contains seven cysteine residues. RT-PCR showed that IL-15 is expressed in many tissues in the rabbit, including heart, spleen, lung, liver, muscle, and kidney. Expressed and purified recombinant RIL-15, in the absence of the 48 aa leader sequence, stimulated the proliferation of cells of the mouse T cell line, CTLL-2, and its activity is comparable to hIL-15. Western blotting demonstrated that recombinant RIL-15 can be recognized by anti-IL-15 neutralization antibody. Western blotting also confirmed that IL-15 is present in many tissues including heart, spleen, lung, liver, muscle, and kidney.