树鼩（Tupaia belangeri）ISG15分子全长克隆及分子生物学功能分析  

Cloning, Structure Prediction and Function Analysis of Complete cD- NA of Tupaia belangeri ISG15

作　　者：夏幼辰[1] 吴珺[1] 高珊[2] 赵西平[3] 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院感染科,武汉市430022 [2]华中科技大学同济医学院附属襄樊医院呼吸内科,湖北省襄樊市441021 [3]华中科技大学同济医学院附属同济医院感染科,武汉市430030

出　　处：《医学分子生物学杂志》2014年第3期132-138,共7页Journal of Medical Molecular Biology

基　　金：国家传染病科技重大专项课题（No.2008ZX10002011,2012ZX10004503）,科技部国际合作与交流专项（No.2011DFA31030）,华中科技大学研究生创新创业基金（No.HF.11.30-2013）

摘　　要：目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基础。方法根据Genbank中灵长类及其他哺乳类动物ISG15分子序列保守区设计引物，使用SmarterRace方法扩增树购ISG15全长序列。在序列两端设计特异性引物，引入酶切位点，进行全长片段扩增。全长PCR产物纯化回收后连接至pMD18-T载体，构建重组质粒pMD18-T-tbISG15。对重组质粒进行酶切鉴定、测序。对序列进行同源性及种系进化，同时使用SWISSMODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树购ISG15全长序列共687bp，编码157个氨基酸。树晌ISG15与其他哺乳动物ISG15高度同源，与人的核苷酸及氨基酸序列同源性分别高达72．99％及71．34％，核苷酸及氨基酸序列进化树分析均显示树嗣种系最为接近灵长类动物。结构域分析提示：树嗣ISG15主要由两个类泛素样结构域构成。软件预测所得树购ISG15三维结构与人ISG15三维结构高度相似，且具有类泛素样三维结构，动力学检测提示本试验预测的树嗣ISG15三维结构稳定，可信度高。结论对树胸ISG15的克隆进一步完善了对树购模型的认识。为进一步在体内研究HCV感染的天然免疫机制奠定了分子生物学基础。Objective We aim to characterize the complete cDNA sequence and the function of Tupaia belangeri ISG15, to offer molecular biology information for the study of HCV in Tupaia belangeri. Material and Methods Primers were designed according to the consensus sequence of mammalian ISG15, and the complete cDNA sequence was cloned by Smarter RACE method. Specific primers including restricted enzyme site were designed for complete cDNA amplifica- tion. PCR product was extracted and purified, and then cloned into the pMD18-Tvector. Positive clones were selected by restricted enzyme digestion and sequenced. Homology analysis and phylogenetic tree were calculated by software, and the secondary and 3-D structures of tupaia ISG15 were predicted by SWISS MODEL software. Results The complete sequence of 687 nucleotides and 157 amino acids of tupaia ISG15 were obtained. The nucleotide and amino acid sequence of tupaia ISG15 and human ISG15 shared a homology of 72.99% and 71.34% separately. Phylogenetic tree indicated that tupaia ISG15 was most related to primate species. SWISS MODEL prediction showed that tupaia ISG15 had two ubiquitin-like domains, and the 3-D structure was similar to human ISG15, Errat and verify3 D evaluation results indicated that the predicated 3-D structure of tupaia ISG15 was stable and reliable. Conclusion Our results improved the understanding of tupaia as an animal model, offered important molecular biology information for the further in vivo study of innate immunity of HCV infection in Tupaia belangeri.

关 键 词：树鼩 丙型病毒性肝炎 动物模型 干扰素刺激基因15 天然免疫 

分 类 号：Q7[生物学—分子生物学] Q93