羊IL-1Ra基因全长cDNA克隆及生物信息学分析

试验旨在对羊白介素1受体颉颃因子（interleukin-1 receptor antagonist，IL-1Ra）基因进行全长cDNA克隆及生物学分析。根据布鲁氏菌感染羊白细胞层SSH cDNA文库中的IL-1Ra基因序列信息及已知核苷酸序列（GenBank登录号：KC425613.1）设计引物，利用RT-PCR结合RACE方法扩增并克隆IL-1Ra基因，对其进行序列及相关分子特性分析。结果表明，羊IL-1Ra基因全长为1 228 bp，可编码174个氨基酸，含有1个完整的IL-1保守结构域和1个IL-1Ra结构域（PHA02651结构域）。IL-1Ra分子质量为19 765.8 u，等电点（pI）为5.72，其分子式为C₈₈₅H₁₃₈₅N₂₃₅O₂₅₆S₁₁，且含有信号肽。二级结构分析显示，IL-1Ra分子存在较多的β折叠及受体结合位点，与三级结构预测结果一致，且与人/鼠IL-1Ra分子的空间结构相似度极高，达到90%以上。羊IL-1Ra有IL-1特有的三叶草结构，其酶结合结构域位于TYR⁴⁷至GLU⁶⁶之间。将羊IL-1Ra与牛、虎鲸、宽吻海豚、猪、家犬、人、鼠等14个物种进行蛋白质同源性比对，发现在各物种间存在5个高度同源的半胱氨酸位点。系统进化树分析发现，羊IL-1Ra基因全长cDNA所编码的氨基酸序列同山羊、牛单独形成一个分支。本研究结果为今后深入研究IL-1Ra基因的生物学功能奠定了基础。

Cloning and Bioinformatics Analysis of IL-1Ra Gene Full-length cDNA in Sheep

This study was aimed to clone ovine interleukin-1 receptor antagonist (IL-1Ra) gene and analyze the gene structure using bioinformatics.The primers was designed according to the obtained SSH cDNA library IL-1Ra gene sequence information and the known nucleotide sequence(GenBank accession No.:KC425613.1),IL-1Ra gene was amplified and cloned by RT-PCR and RACE methods,IL-1Ra sequence and its related molecular characteristics were analyzed.The results showed that the length of IL-1Ra gene was 1 228 bp,and encoded 174 amino acids.It was predicted that IL-1Ra had a complete IL-1 conserved domain and IL-1Ra domain (PHA02651 domain).The molecular weight of IL-1Ra was 19 765.8 u,the isoelectric point (pI) was 5.72,its molecular was C₈₈₅H₁₃₈₅N₂₃₅O₂₅₆S₁₁.IL-1Ra contained the signal peptide.The secondary structure of IL-1Ra had more β-fold and receptor binding sites,which were consistent with the predicted results of tertiary structure,and had a similarity to the spatial structure of human/mouse IL-1Ra,up to more than 90%.The IL-1Ra contained a clover structure which was peculiar to the IL-1,and its enzyme-binding domain was located between TYR⁴⁷ and GLU⁶⁶.The results showed that there were five highly homologous cysteines between sheep and the other fourteen species.By establishing the phylogenetic tree of IL-1Ra,it was found that the full-length cDNA of IL-1Ra in sheep formed a branch with goat and cattle.This study result could provide the basis for the further study of the biological function of IL-1Ra gene.