松江鲈白介素15(TfIL-15)的结构特征与重组表达

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

为研究白介素15(Interleukin-15, IL-15)在松江鲈(Trachidermus fasciatus Heckel)先天免疫中的功能,本研究利用RACE技术克隆得到松江鲈IL-15基因(命名为TfIL-15)的全长cDNA序列,其长度为1140bp,包括5'-非编码区(5'-UTR) 165bp、开放阅读框(ORF) 522bp和3'UTR 453bp。在5'UTR区域,存在4个读码框外的AUG翻译起始位点。基因ORF编码173个氨基酸(aa),其中,前59aa为信号肽序列。成熟肽全长为114aa,预测分子量为12.975kDa,理论等电点为5.15。同源比对发现,鱼类IL-15变异程度较高,TfIL-15与其他鱼类IL-15同源性在23%~61%之间。多序列比对和三维结构构建结果显示,TfIL-15具有典型4个α螺旋二级结构,形成二硫键的4个半胱氨酸高度保守。qRT-PCR分析表明,TfIL-15广泛表达于松江鲈各组织中。腹腔注射脂多糖(Lipopolysaccharides, LPS)后,TfIL-15 mRNA在血液、皮肤、肝脏和脾脏中均上调表达。在皮肤和血液中,刺激后2h表达量迅速上调至最高峰,分别为对照组的74倍和41倍。脾脏和肝脏在刺激后12h分别达到对照组的3倍和18倍。肝脏中,刺激后96h,表达量再次上调至对照组的86倍。上述结果表明,TfIL-15可能参与了松江鲈抵抗外界刺激的先天免疫过程。另外,通过构建TfIL-15成熟肽的原核表达载体,成功获得重组蛋白,为进一步研究TfIL-15蛋白的功能奠定了基础。

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.