梅花鹿GAL-1基因编码区克隆及表达检测

摘要

The Galectin-1 (GAL-1) gene in the deer antler stem cells is found to be highly expressed during antler regeneration period,indicating that it may be an important regulator for antler regeneration.To study the expression and possible biological function of GAL-1 in antler stem cells,antlerogenic periosteum and pedicle periosteum(potentiated pedicle periosteum and dormant pedicle periosteum) and facial periosteum of sika deer (Cervus nippon) were used as experimental materials.In this study,the GAL-1 gene CDS region of sika deer was cloned and bioinformatics analysis of GAL-1 was carried out using software or website.The relative expression of GAL-1 in the antler stem cells was detected by using Western-blot and qRT-PCR technique.The results showed that the GAL-1 ORF was 408 bp in length and encoded 134 amino acids.The relative molecular mass was 14.69 kD and the amino acid sequence was close to those of bovine (Bos taurus)and Ovis aries.GAL-1 was mainly distributed in the interstitial substance.It does not have transmembrane regions and glycosylation sites,and there may be two phosphorylation sites.The secondary structure of the GAL-1 protein was mainly β-sheets and random coil,and the tertiary structure was similar to that of the bovine.Expression level of GAL-1 was higher in the facial periosteum.In this study,the structural characteristics of GAL-1 protein was predicted,the expression level of GAL-1 protein in antler stem cells was detected and would provide the basic data for the further study of antler regeneration.%鹿茸干细胞中半乳糖凝集素1(galectin-1,GAL-1)基因被发现高表达,推测其是鹿茸再生中的一个重要调控因子.为研究GAL-1在鹿茸干细胞中的表达及可能的生物学功能,本研究以东北梅花鹿(Cervus nippon)鹿茸的干细胞组织,即生茸区骨膜和角柄骨膜(致敏角柄骨膜与休眠角柄骨膜)以及对照组织面部骨膜为材料,克隆了梅花鹿的GAL-1基因CDS区序列,并对该序列进行生物信息学分析;利用Western-blot及qRT-PCR技术检测了GAL-1生鹿茸干细胞中的相对表达量.结果显示:梅花鹿GAL-1ORF全长为408 bp(GenBank No.MG882483),编码134个氨基酸;相对分子质量为14.69 kD,氨基酸序列与牛(Bos taurus)、羊(Ovis aries)高度接近;主要定位于细胞外,无跨膜区,无糖基化位点,有两个磷酸化位点,二级结构主要为β-折叠与无规卷曲;三级结构与牛的三级结构相似;GAL-1在面部骨膜中表达量更高.本研究预测了梅花鹿GAL-1蛋白的结构特点,检测了GAL-1蛋白在鹿茸干细胞中的表达情况,为进一步研究鹿茸再生提供数据支持.