狐、貉和水貂犬瘟热病毒受体SLAM的基因克隆及其真核表达

Molecular cloning and eukaryotic expression of Canine distemper virus receptor gene SLAM of fox, raccoon dog and mink

信号淋巴激活分子(SLAM)为犬瘟热病毒(CDV)感染其宿主动物识别的细胞受体。本试验应用RT-PCR从狐狸、貉和水貂的外周血淋巴细胞中克隆到其相应SLAM基因。基因测序比较发现,狐狸、貉与同科的犬SLAM基因编码区长度均为1029bp,核苷酸同源性高于98.6%;而水貂SLAM基因编码区长度为1020bp,与以上三种动物遗传关系较远(核苷酸同源性<83.7%),但与海豹SLAM基因遗传关系较近(核苷酸同源性为91.4%)。基于不同动物SLAM基因序列的系统进化树分析显示,犬、狐狸、貉、水貂和海豹在进化树上构成了以CDV为感染宿主的遗传分支。氨基酸序列比较显示,该5种动物SLAM分子上均存在一个长度为26个氨基酸的信号肽序列,且在空间结构上影响宿主——病毒特异性的8个关键氨基酸均完全保守。通过构建表达该狐狸、貉、水貂SLAM基因的三种真核表达质粒,分别转染CRFK细胞后,应用CDV强毒感染试验证实,CDV均能在三种转染细胞上产生明显的细胞病变效应(CPE),而未转染CRFK细胞对照无CPE产生,由此证实作为CDV细胞受体的狐、貉和水貂的SLAM,体外表达后能明显增强犬瘟热强毒株对非敏感细胞的感染能力。

Signaling lymphocyte activation molecule （SLAM） is one of the cellular receptors for Canine distemper virus （CDV） infection. In this study, we first cloned and sequenced the SLAM genes from peripheral blood lymphocytes of fox （Vulpes vulpes）, raccoon dog （Nyctereutes procyonoides） and mink （Mustela lutreola） respectively. Identical with the dog （Canine） SLAM, SLAM cDNAs both of fox and raccoon dog were 1 029 bp length encoding 342 amino acids. The nucleotide sequence homologies of SLAMs among fox, raccoon dog and dog were highly homologous （98.6%）.The SLAM cDNA of mink was 1 020 bp in length encoding a polypeptide of 339 amino acids and had high homology with spotted seal （Phoca largha） SLAM （91.4% at nt level, 87.9% at aa level）.Phylogenetic analysis based on the nucleotide sequences of SLAM genes of different species showed the natural hosts including dog, fox, raccoon dog, mink and seal for CDV formed a monophyletic cluster. In addition to the 26 amino acid residue predicted signal peptide of SLAMs, there were 8 amino acid residues in the V region of SLAM thought to be involved in determining host-virus specificity were completely conserved, irrespective of the species of CDV infection. We also established three eukaryotic recombinant plasmids using an pIRES2-EGFP vector which expressed the SLAM cDNAs of fox, raccoon dog and mink in transient transfection CRFK cells. One day after inoculation with CDV virulent strain, all the SLAM-expressing CRFK cells produced extensive cytopathic effects （CPE）.Our results suggest that CDV use SLAMs of fox, raccoon dog and mink as a cellular receptor.