Cloning and expression of rabbit interleukin-15.
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.
National Cancer Institute-Frederick, National Institutes of Health, Frederick, MD, USA.
Changyun
C
Xiong
Patricia M
PM
Hixson
Leonardo H
LH
Mendoza
C Wayne
CW
Smith
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D019409:Interleukin-15 / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits / Q000235:genetics* / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014018:Tissue Distribution
D018076:DNA, Complementary; D019409:Interleukin-15; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins