Characterization and Recombinant Protein Expression of Interleukin-15 from Roughskin Sculpin, Trachidermus fasciatus

Abstract:

Interleukin 15 (IL-15) is an important cytokine of fish immune system. In the present study, the IL-15 cDNA named as TfIL-15 was cloned from roughskin sculpin, Trachidermus fasciatus. The full-length of TfIL-15 cDNA is 1140 bp, which contains a 5¢-UTR (untranslated region) of 165 bp, a 3¢-UTR of 453 bp and an open reading frame (ORF) of 522 bp, encoding a polypeptide of 173 amino acids (aa) with a putative 59 aa-long signal peptide. Four out-of-frame AUG initiation codons, the negative translational regulators of mammalian IL-15 genes were also detected in the 5¢-UTR of TfIL-15. The protein sequence shared 23%~61% identity with reported fish IL-15s, displaying relatively high degree of variation. TfIL-15 homologues also contained four highly conserved cysteine residues allowing the formation of two disulfide bridges along with four predicted α-helices. Phylogenetic analysis grouped roughskin sculpin with other fish on a separated branch, excluded from mammalian and avian IL-15s. Quantitative real-time PCR (qRT-PCR) analysis showed that TfIL-15 was widely expressed in all detected tissues, with the highest expression in the heart. Post LPS challenge, TfIL-15 increased rapidly to the maximum of 74 folds and 41 folds compared with that of the control group at 2 h post challenge (hpc) in the skin and blood. The induction of TfIL-15 mRNA in the spleen and liver was 3 folds and 18 folds at 12 hpc. Interestingly, at 96 hpc, the expression of TfIL-15 in the liver was up-regulated again to the 86 folds higher than that of the control group. These results indicate that TfIL-15 may play an important role in fish innate immune response against microbial infections. Furthermore, the mature peptide of TfIL-15 was expressed in E. coli BL21 (DE3) cells successfully, laying a foundation for further research on the function of TfIL-15 protein.