比格犬SLAM基因的克隆与序列分析

Cloning and Sequence Analysis of Signaling Lymphocyte Activation Molecule Gene in Beagle Dog

对比格犬的犬瘟热病毒细胞受体——信号淋巴细胞激活因子(SLAM)基因进行克隆及测序分析,并与其他物种的SLAM受体进行比较,以期发现其关键结构部分。用RT-PCR从Con A刺激后的犬外周血淋巴细胞中扩增SLAM基因,将目的片段连接到PMD18-T载体上,转化感受态细胞,筛选阳性克隆,测序。结果显示,克隆的比格犬SLAM基因序列全长1 029bp,编码342aa。与人、牛、狐、羊和海豹等SLAM序列同源性约74.6%～98.4%。SLAM基因序列的系统进化树分析显示,犬、狐狸和海豹的关系较近。理化分析显示SLAM属于具有信号肽的Ⅰ型跨膜糖蛋白,信号肽位于N段1aa～26aa,跨膜区位于239aa～263aa,膜外区存在5个潜在的糖基化位点,膜内区包括3个保守的酪氨酸基序。SLAM基因部分区域的高度保守性,为犬瘟热病毒的宿主范围扩大奠定了基础。

In order to find critical structural characteristics of cellular receptors of canine distemper virus,the gene sequences of signaling lymphocyte activation molecules（SLAM） of beagles were cloned,sequenced and analyzed.The cDNA sequences were amplified from peripheral blood lymphocytes,by stimulation with Con A,of the beagles.The target genes were connected to the PMD-18 vector,and then transformed to competent cells.The positive clones were sequenced.The result showed that the nucleotide（nt） open reading frame sequence of SLAM gene in beagles was 1 029 nucleotides in length encoding a polypeptide of 342 amino acids.Sequence analysis revealed 74.6%-98.4% identities among the six species at the nucleotides level.Phylogenetic relationship based on the amino acid sequences of SLAM protein revealed that canine,fox and seal fall under a defined cluster.The physicochemical properties analysis proved that the SLAM is a glycoprotein with signal peptide N-terminal 1 aa-26 aa,and possessed transmembrane domain in 239 aa to 263 aa.The SLAM has five potential glycosylation sites in extracellular domain and three tyrosine motifs in intracellular region.SLAM has high homology in those species,and this lay a foundation in enlarging of host range of canine distemper virus.