Cloning of cDNA sequence and expression characteristics analysis of ISG15 gene in Mauremys mutica

【Objective】To clone the interferon-stimulated gene 15（ISG15）in Mauremys mutica，analyze its expression characteristics，which could provide theoretical basis for exploring the immune function and mechanism of ISG15 protein during the invasion of disease-resistant microorganism in M. mutica.【Method】Using RACE technology，the fulllength cDNA sequence of the ISG15 gene from M. mutica（MmISG15）was cloned. Bioinformatics analysis was conducted，and the expression of MmISG15 gene in various tissues of healthy M. mutica was assessed using real-time fluorescence quantitative PCR. Additionally，the expression changes of the MmISG15 gene were monitored following stimulation with lipopolysaccharide（LPS），polycytidylic acid［Poly（I∶C）］and Klebsiella pneumoniae，with PBS serving as the control.【Result】The full-length cDNA sequence of MmISG15 gene was 1276 bp，including 5' untranslated regions （5'-UTR）and 3' untranslated regions（3'-UTR）of 126 and 650 bp respectively，and an open reading frame（ORF）of 498 bp，encoding 165 amino acid residues with protein molecular mass and isoelectric point（pI）of 15.3 kD and 7.70 respectively. The N-terminus of MmISG15 protein contained 2 ubiquitin-like domains（UBD），and the C-terminus contained a highly conserved LRLRGG sequence. Homology analysis revealed that the amino acid sequence of MmISG15 shared 55.57%-95.54% similarity with ISG15 amino acid sequence of other reptiles，and 34.09%-49.02% and 38.64%- 48.25% similarity with mammals and fish respectively. Phylogenetic analysis showed that MmISG15 clustered with ISG15 from other reptiles，and the most closely related to turtles（Mauremys reevesii）. Real-time fluorescence quantitative PCR results indicated that MmISG15 gene was expressed in all 8 examined tissues of healthy M. mutica，with the highest relative expression in the spleen，followed by liver. Lower relative expressions were observed in the foregut，hindgut，kidney，lung and muscle tissues，with the lowest in brain tissue. Following LPS stimulation，there was no significant difference in the relative expression of MmISG15 gene in the spleen and liver tissues compared to the control（P>0.05）. Post-stimulation with Poly（I∶C），extremly significant up-regulation of MmISG15 gene was observed at 6 h in the spleen and liver tissues（P<0.01，the same below），reaching a peak at 12 h. Following stimulation with K. pneumoniae，extremely significant up-regulation in MmISG15 gene expression was noted at 12 h in the spleen and liver tissues，reaching the highest level at 48 h post-stimulation.【Conclusion】MmISG15 is a classical ubiquitin-like protein，MmISG15 gene primarily expresses in the spleen and liver tissues of M. mutica. Stimulation with Poly（I∶C）and K. pneumoniae upregulates the expression levels of the MmISG15 gene in spleen and liver，indicating that MmISG15 gene plays an important role in the immune response of M. mutica against virus and bacterial invasion，with its encoded protein playing important immunological role.