树鼩CD4全长编码序列的克隆及分子特征分析

Cloning of full-length coding sequence of tree shrew CD4 and prediction of its molecular characteristics

树鼩作为多种人类疾病研究模型的可能性已受到广泛关注,但尚缺乏研究其免疫功能的基本标志以及单克隆抗体。该实验首先以树鼩外周血总RNA为材料,通过RT-PCR扩增得到长度为1365bp的树鼩CD4全长编码序列,并确定了数据库中缺失的两个片段,进而通过ClustalW等软件对其序列和分子特征进行分析,发现树鼩CD4氨基酸序列胞外和胞内域保守性较好,且与人类和猴的亲缘关系较近。虽然树鼩和人CD4分子表面大部分区域均带正电荷,但与人CD4胞外域D1相比,树鼩CD4D1结构区域表面带负电荷较多,且多出两个N-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CD4单克隆抗体制备及功能研究奠定了基础。

The tree shrews,as an ideal animal model receiving extensive attentions to human disease research,demands essential research tools,in particular cellular markers and monoclonal antibodies for immunological studies.In this paper,a 1 365 bp of the full-length CD4 cDNA encoding sequence was cloned from total RNA in peripheral blood of tree shrews,the sequence completes two unknown fragment gaps of tree shrews predicted CD4 cDNA in the GenBank database,and its molecular characteristics were analyzed compared with other mammals by using biology software such as Clustal W2.0 and so forth.The results showed that the extracellular and intracellular domains of tree shrews CD4 amino acid sequence are conserved.The tree shrews CD4 amino acid sequence showed a close genetic relationship with Homo sapiens and Macaca mulatta.Most regions of the tree shrews CD4 molecule surface showed positive charges as humans.However,compared with CD4 extracellular domain D1 of human,CD4 D1 surface of tree shrews showed more negative charges,and more two N-glycosylation sites,which may affect antibody binding.This study provides a theoretical basis for the preparation and functional studies of CD4 monoclonal antibody.