番鸭源呼肠孤病毒YJL株σNS基因克隆和序列分析

Cloning and sequence analysis of σNS gene of reoviruses YJL strain isolated from muscovy duck

参考GenBank上公布的禽呼肠孤病毒(ARV)和番鸭呼肠孤病毒(MDRV)σ非结构基因(σNS)序列设计合成一对引物,对番鸭源呼肠孤病毒YJL株σNS全基因进行RT-PCR扩增,克隆到pMD18-T载体中,并对克隆产物进行PCR鉴定和测序;YJL株σNS的编码基因位于S4节段,基因全长1192 bp,其5′端和3′端序列分别为5′-GCTTTT和3′-TATTCATC,具有典型的禽正呼肠孤病毒基因末端碱基序列;σNS基因只有一个有效阅读框(24-1127 bp),编码由367个氨基酸组成的σNS蛋白;σNS蛋白的分子质量约为40.57 ku,等电点(PI)为7.875.YJL株与法国MDRV-89026株σNS基因核苷酸的同源性为77.8%,推导氨基酸的同源性为90.2%;与ARV-S1133株σNS基因的同源性为99.4%,推导氨基酸的同源性为99.5%;系统进化树分析表明,YJL株σNS基因和蛋白与S1133株的亲缘关系更近,处在ARV分支上.可见,番鸭源呼肠孤病毒YJL株属于ARV,同时也表明我国发病番鸭群中存在不同基因群的呼肠孤病毒感染.

σNS genes of muscovy duck reovirns YJS were amplified by RT-PCR with the primers designed by the reported avian and muscovy duck reovirnses.The PCR products were cloned to the pMD18-T vector and identified by PCR analysis and sequencing.The sequence results showed that σNS genes are 1 192 bp.The σNS conserved 5′ and 3′ terminal motifs of YJS are 5′-CCTTTTT and TCATC-3′,which are the same as that of avian reovirnses.It contains one open reading frames that encodes a protein of 367 amino acids with a molecular mass of 40.57 ku and an isoelectric point of 7.875.The homology of σNS gene and deduced amino acid sequence between YJS and muscovy duck reoviruse 89026 are 77.8% and 90.2%,compared to that of avian arthritis virus S1133,the homology are 99.4% and 99.5% respectively.The phylogenetic analysis showed that the sigma NS genes of YJL and ARV-S1133 fell into the same cluster and belonged to the cluster of avian reoviruses,which demonstrated that sigma NS encoding gene of YJL,which was isolated from muscovy duck,was closely related to avain reovirus.The results of this study indicated that several kinds of gene groups avain and duck reovirus exit in the muscovy duck in south China,which suggested that there are avain reovirus multiple infections in the muscovy duck,simultaneously explained that avain reovirus distributed complexity and the multiplicity in China.