鸭Toll样受体3基因克隆及序列分析

Cloning and Sequence Analysis of TLR3 Gene in Peking Duck

为研究鸭Toll样受体3(TLR3)的结构及功能,根据GenBank中已公布的番鸭TLR3(JQ910167.1)基因设计引物,采用RT-PCR从北京鸭外周血单个核细胞中克隆出北京鸭TLR3,命名为duTLR3。利用生物信息学技术预测其结构及功能,研究表明,duTLR3基因开放阅读框为2 688bp,编码895个氨基酸,富含17.0%亮氨酸;该分子属于Ⅰ型跨膜受体,由胞外区(1-695aa)、跨膜区(696-718aa)和胞内区(719-895aa)3部分组成,N端含有一个信号肽序列,胞外富含18个亮氨酸重复序列(LRR),胞内含有Toll/IL-1R同源区结构域。核苷酸序列同源性分析显示,duTLR3与金定鸭TLR3亲缘关系最接近达到99.6%;与番鸭、鹅、原鸡、斑胸草雀的亲缘关系次之,并同属于禽类分支上;与番鸭、鹅、原鸡、斑胸草雀、人、猕猴、狒狒、小鼠、大鼠、猪、牛、羊、猫亲缘关系渐远;与草鱼和鲤鱼亲缘关系最远。北京鸭体内组织分布结果显示,duTLR3为组成性表达。本研究成功克隆了北京鸭TLR3基因,预测分析并证实了duTLR3具有典型的TLR家族结构特征,为后期探究TLR3如何识别病毒的RNA及引起下游信号级联反应奠定了基础。

To research the structure and function of TLR3 in Peking duck, according to the published Cairina moschata TLR3 gene sequence in the GenBank（JQ910167.1）,the primers were designed. Peking duck TLR3 （named duTLR3） gene was cloned from peripheral blood mononuclear cell of duck by using RT-PCR, and the structure and function were predicted by utilizing bioinformaties technology. The results of sequence analysis showed that the length of cloned TLR; eDNA is 2688 bp, including a complete open reading frame （ORF）, it encodes a peptide of 895 amino acids, which is rich of 17.0% leucine. Toll-like receptor 3（duTLR3） is type I transmembrane receptor, consisting of an extracellular domain （1-695 aa）, a transmembrane domain （696-718 aa） and an intracellular domain（719-895 aa）. Furthermore there is a signal peptide in its N terminal besides its extracellular region with 18 leucine rich repeats（LRR） while its intracellular region has a Toll/interleukin-1 receptor（TIR） domain. Nucleotide sequence homology analysis showed that Peking duck has higher homology with Anas latyrhynchos （XM- 005008981.1） up to 99.6%, the homology of nucleotide sequence of duTLR3 with those of Cairina moschata, Anser anser , Callus, Taeniopygia guttata , is much higher than that of Homo sapiens. Macaca mulatta , Papio Anubis , Mus musculus , Rattus norvegicus , Sus scro f a , Bos taurus, （Avis aries, Felis catus , Ctenopharyngodon idella and Cyprinus carpio being the lower homology than duTLR3, duTLR3 is constitutively expressed in all tissues of Peking duck. The results showed that duTLR3 gene was cloned successfully and was predicted with characteristics of typical structure of TLR family. It laied a solid virus and activates downstream signaling cascades. foundation on researching about how TLR3 recognizes