番鸭呼肠孤病毒非结构基因的克隆和序列分析

参考GenBank禽呼肠孤病毒(Avian Reovirus,ARV)和番鸭呼肠孤病毒(Muscovy Duck Reovirus,MDRV)非结构基因(NS)序列设计合成一对引物,对番鸭呼肠孤病毒S14和C4株NS基因进行RT-PCR扩增,克隆到pMD18-T载体中,并对克隆产物进行酶切鉴定和测序;番鸭呼肠孤病毒NS基因由1 291 bp核苷酸组成,与禽呼肠孤病毒NS基因相比,在非编码区第1155位少一个碱基,本文第一次证实1291bp是番鸭呼肠孤病毒NS基因特有的长度;番鸭呼肠孤病毒S14和C4株NS基因的5'末端和3'末端分别为5‘GCTTTT和TCATC-3',是禽类呼肠孤病毒基因末端特有的碱基序列,S14和C4株NS基因的的有效阅读框(24～1127bp)编码367个氨基酸组成的蛋白,分子量约为40kDa;番鸭呼肠孤病毒S14和C4株NS蛋白等电点分别是7.3和7.0,GC含量分别为54.26%和53.71%,番鸭呼肠孤病毒S14和C4株NS基因间核苷酸同源性为99.3%,仅有4个氨基酸差异,S14和C4与法国番鸭呼肠孤病毒89026株NS基因核苷酸同源性分别为87.8%和87.9%,与鸡关节炎病毒S1133 NS基因同源性分别为79.0%和79.3%;进化树分析表明本研究中的两株番鸭呼肠孤病毒非结构基因(NS)与番鸭呼肠孤病毒的亲缘关系比禽呼肠孤病毒近的多,建议番鸭呼肠孤病毒应归属为正呼肠孤病毒属第二个亚群中不同于禽和内尔森贝海湾呼肠病毒独立基因群.

Cloning and sequence analysis of muscovy duck reoviruses S14 and C4L8 strains NS gene

NS genes of muscovy duck reovirus S14 and C4 were amplified by RT_PCR with the primers designed by the reported avian and muscovy duck reoviruses. The PCR products were cloned to the pMD18_T vector and identified by enzyme cutting analysis. The sequence results showed that both NS genes are 1 191 bp. The NS conserved 5' and 3' terminal motifs of S14 and C4 are 5'_GCTTTTT and TCATC_3', which are the same as that of avian reoviruses. It contains one open reading frame that encodes a protein of 367 amino acids with a molecular mass of 40.0 kDa. Phylogenetic analysis shows that NS gene of S14,C4 and 89026 are all grouped with avian reoviruses. The homology of NS gene between S14 and C4 is 99.3 %. The NS genes of S14 and C4 are related to that of muscovy duck reoviruse 89026, respectively with 87.8 % and 87.9% homology. Compared to that of avian arthritis virus S1133,the homology of NS gene of S14 and C4 are respectively 79.0 % and 79.3 %. Generally, relationship of muscovy duck reovirus S14 and C4 to 89026 are more similar than that to avian arthritis virus S1133. The relationship of NS genes of muscovy duck S14 and C4 are more related to 89026 than to avian reovirus. The results of this study give the suggestion that the muscovy duck reoviruses might belong to one special gene group among avian reovirus of second subgroup of orthoreovirus genus.