While the tree shrew (Tupaia belangeri chinensis) is an excellent animal model for studying the mechanisms of human diseases, but few studies examine interleukin-2 (IL-2), an important immune factor in disease model evaluation. In this study, a 465 bp of the full-length IL-2 cDNA encoding sequence was cloned from the RNA of tree shrew spleen lymphocytes, which were then cultivated and stimulated with ConA (concanavalin). Clustal W 2.0 was used to compare and analyze the sequence and molecular characteristics, and establish the similarity of the overall structure of IL-2 between tree shrews and other mammals. The homology of the IL-2 nucleotide sequence between tree shrews and humans was 93%, and the amino acid homology was 80%. The phylogenetic tree results, derived through the Neighbour-Joining method using MEGA5.0, indicated a close genetic relationship between tree shrews, Homo sapiens, and Macaca mulatta. The three-dimensional structure analysis showed that the surface charges in most regions of tree shrew IL-2 were similar to between tree shrews and humans; however, the N-glycosylation sites and local structures were different, which may affect antibody binding. These results provide a fundamental basis for the future study of IL-2 monoclonal antibody in tree shrews, thereby improving their utility as a model.
树鼩作为多种人类疾病模型已受到广泛关注, 而免疫因子对于树鼩模型评价至关重要, 但目前对其白细胞介素-2(IL-2)的研究鲜有报道。该实验以经ConA (concanavalin)诱导培养的树鼩淋巴细胞总RNA为模板, RT-PCR克隆出465 bp的树鼩IL-2全长编码序列, 并采用Clustal W软件分析其序列和分子特征。结果表明树鼩IL-2 cDNA编码一个由154个氨基酸组成的蛋白质, 其cDNA及氨基酸序列与人的同源性分别为93%及80%, 且其整体结构与人IL-2相似。MEGA5.0软件构建的进化树表明, 树鼩与人及恒河猴的亲缘关系较近。Pymol软件对树鼩和人IL-2氨基酸序列进行的三维结构模建表明, 两者的IL-2分子三维空间结构基本相似, 表面大部分区域所带电荷相同, 但在某些区域差异较大, 且树鼩多出一个糖基化位点, 这些差异对抗体的结合可能存在影响。该研究为今后树鼩IL-2单克隆抗体的制备及功能研究奠定了基础。
Keywords: Cloning; Function; Interleukin 2; Structure; Tree shrews.