禽白血病病毒贵州流行株env基因克隆与序列分析

为了解禽白血病病毒（ALV）贵州流行株的遗传变异情况及分子特征，本试验基于ALV env基因设计合成引物对禽白血病贵州临床病例进行目的基因扩增、克隆和序列分析。结果显示，从临床病例中筛选获得3份阳性样本，PCR扩增均获得大小约921 bp的目的基因片段，将其命名为：GZ-ALV-1株、GZ-ALV-2株和GZ-ALV-3株。序列分析结果显示，3株ALV贵州流行株之间核苷酸同源性在97.2%~97.6%之间，与国内外ALV-J的同源性相对较高，为93.1%~99.3%；而与A、B、C、D、E、K亚群ALV同源性仅为51.4%~53.2%。系统进化分析显示，3株ALV贵州流行株与ALV-J亚群参考株处于同一分支，表明本试验所检测的ALV毒株均为ALV-J亚群；与A、B、C、D、E、K亚群处于不同进化分支。基因变异分析显示，3株流行株37处相同核苷酸变异导致17处氨基酸发生位点变异，其中9个可变点在高变区hr1和hr2，1个可变点在低变区vr3。结果表明，3株ALV贵州流行株均为ALV-J亚群，env基因存在位点发生了变异，且可变位点位于序列高变区。本研究结果为明确贵州禽白血病流行概况及ALV的防控与净化提供基础数据。

Cloning and Sequence Analysis of env Gene of ALV Guizhou Strain

In order to understand the genetic variation and molecular characteristics of avian leukaemia virus (ALV) endemic strains in Guizhou,this study designed and synthesized primers based on ALV env gene for the purpose of gene amplification,cloning and sequence analysis of avian leukaemia clinical cases.The results showed that 3 positive samples were selected from clinical cases.The target gene fragments with a size of 921 bp were obtained by PCR amplification and named as GZ-ALV-1,GZ-ALV-2 and GZ-ALV-3 strains.Sequence analysis results showed that the nucleotide homology of 3 ALV epidemic strains in Guizhou ranged from 97.2% to 97.6%,which was relatively high with ALV-J at home and abroad,ranging from 93.1% to 99.3%,while the homology with ALV subgroups A,B,C,D,E and K was only 51.4% to 53.2%.The phylogenetic tree analysis showed that 3 ALV epidemic strains in Guizhou were in the same branch as the reference strains of ALV-J subgroup,which further indicated that all the ALV strains detected in this paper were ALV-J subgroup,while in different branches compared with A,B,C,D,E and K subgroups.The gene mutation analysis showed that 37 same nucleotide mutations of 3 epidemic strains resulted in 17 amino acid mutation sites,including 9 variable points in the high-variable region of hr1 and hr2,and 1 variable point in the low-variable region of vr3.The results showed that 3 ALV strains in Guizhou were ALV-J subgroup,and env gene loci were mutated,and the variable loci were located in the high-variable region.The results provided basic data for the epidemic situation of avian leukemia and the prevention and control and purification of ALV in Guizhou province.