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

摘要

目的：对丙型病毒性肝炎（ hepatitis C virus ， HCV ）小动物模型树鼩的干扰素刺激基因15（ in-terferon stimulated gene 15， ISG15）分子的全长cDNA序列进行克隆及分子生物学功能分析，为树鼩模型在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Smarter Race方法扩增树鼩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 SWISS MODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树鼩ISG15全长序列共687 bp，编码157个氨基酸。树鼩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鼩种系最为接近灵长类动物。结构域分析提示：树鼩ISG15主要由两个类泛素样结构域构成。软件预测所得树鼩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鼩ISG15三维结构稳定，可信度高。结论对树鼩ISG15的克隆进一步完善了对树鼩模型的认识。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 be-langeri.Material and Methods Primers were designed according to the consensus sequence of mammalian ISG15, and the complete cDNA sequence was cloned by Smarter RACE meth-od.Specific primers including restricted enzyme site were designed for complete cDNA amplifica-tion.PCR product was extracted and purified , and then cloned into the pMD 18-Tvector.Positive clones were selected by restricted enzyme digestion and sequenced .Homology analysis and phyloge-netic 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 ISG 15 were obtained.The nucleotide and amino acid sequence of tupaia ISG 15 and human ISG15 shared a homology of 72.99%and 71.34%separately.Phylogenetic tree indica-ted that tupaia ISG15 was most related to primate species .SWISS MODEL prediction showed that tu-paia ISG15 had two ubiquitin-like domains, and the 3-D structure was similar to human ISG15, Er-rat 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 immu-nity of HCV infection in Tupaia belangeri .