金堂黑山羊白介素10基因的克隆及表达分析

为探究山羊白介素10（interleukin 10，IL-10）基因的序列特征及在组织中的相对表达情况，试验采用PCR法扩增并克隆金堂黑山羊IL-10基因，用DNAMAN、DNAStar、ExPASy等生物信息学软件进行序列分析，运用实时荧光定量PCR法检测IL-10基因在金堂黑山羊不同组织中的表达情况。结果显示，金堂黑山羊IL-10基因序列长为379 bp，开放阅读框为276 bp，编码91个氨基酸残基。IL-10蛋白二级结构中含有α-螺旋、β-转角和无规则卷曲，分别为76.92%、2.20%和20.88%。IL-10蛋白三级结构模型与人IL-10（1ilk.1.A）基因同源性最高（86.81%）。系统进化分析结果发现，金堂黑山羊IL-10基因与挪威山羊IL-10基因亲缘关系最近。实时荧光定量PCR检测发现，在金堂黑山羊肺脏中IL-10基因表达水平显著高于脾脏、心脏、肌肉和肾脏（P<0.05），在肾脏中IL-10基因表达水平显著高于脾脏、心脏和肌肉（P<0.05），在脾脏、心脏和肌肉中IL-10基因表达水平较低，但差异不显著（P>0.05）。本试验结果揭示了IL-10基因具有高度的保守性，可能参与调控山羊的免疫应答反应。

Cloning and Expression Analysis of Interleukin 10 Gene in Jintang Black Goat

This study was aimed to explore the sequence characteristics of interleukin 10 (IL-10) gene and its relative expression of different tissues in goat.IL-10 gene of Jintang Black goat was amplified and cloned by PCR,the gene sequence was analyzed by bioinformatics softwares such as DNAMAN,DNAStar and ExPASy,then the expression of IL-10 gene in different tissues was detected by Real-time quantitative PCR.The results showed that the sequence of IL-10 gene was 379 bp,and the open reading frame was 276 bp,encoding 91 amino acid residues.The secondary structure of IL-10 protein contained alpha helix(76.92%),beta turn(2.20%) and random coil(20.88%).The tertiary structure model of IL-10 protein had the highest homology with human IL-10 (1ilk.1.A) gene (86.81%).In the phylogenetic tree,IL-10 gene in Jintang Black goat was closely related to Norwegian goat.Real-time quantitative PCR result showed that the expression level of IL-10 gene in lung of Jintang Black goat was significantly higher than that in spleen,heart,muscle and kidney (P<0.05),the expression of IL-10 gene in kidney was significantly higher than that in spleen,heart and muscle (P<0.05),the expressions of IL-10 gene in spleen,heart and muscle were lower,but there was no significant difference (P>0.05).It revealed that IL-10 gene was highly conserved,and it might be involved in regulating the immune response of goats.