鸭圆环病毒基因组序列分析及其C1截短基因的原核表达

本试验参照GenBank登录的鸭圆环病毒(DuCV)基因组序列,设计2对DuCV特异性引物,运用PCR方法从病死番鸭法氏囊中分段扩增出DuCV LJ07株基因组,将扩增片段分别克隆获得重组质粒,测序后得到全长为1 995nt的DuCV LJ07株全基因组序列,与GenBank登录的DuCV基因组序列的同源性迭83.6%～96.5%.经基因结构分析发现,DuCV LJ07株的基因组有6个开放阅读框(ORF),其中V1、C1是2个最主要的ORF,分别编码Rep蛋白和Cap蛋白;在V1和C1的5'起始端之间有与启动滚环复制有关的一茎环结构和2个正向重复序列.本试验同时还构建了去除核定位信号的C1截短基因的原核表达载体,诱导表达后经SDS-PAGE和Western-blotting分析表明C1截短基因已成功地在大肠杆菌中表达出Cap截短蛋白,为建立DuCV抗体血清学检测方法和开展DuCV感染的血清学调查奠定了基础.

Genomic sequence analysis of duck circovirus and prokaryotic expression of its C1 truncated gene

The whole genome of duck circovirus(DuCV) LJ07 strain from bursa of dead muscovy ducks was amplified by PCR based on two pairs of specific primers designed according to DuCV genome published.The whole genome of DuCV LJ07 strain was 1 995 nt in size,which shared high identities(83.6%-96.5%) with DuCVs published in GenBank.The analysis results of the whole genome showed it contains the six open reading frames(ORFs),and V1 and C1 of them,encoding Rep protein and Cap protein,respectively were the two major genes.One stem-loop structure and two directly repeat sequence elements,which were indicative of a rolling-circle mode of replication,located at the intergenic regions of the 5′-end of V1 and C1.The C1(absent the sequence of nuclear localization signal) was then subcoloned into the expression vector pGex-6p-1 and expressed efficiently in E.coli expression system by the detections of SDS-PAGE and Western-blotting.This study laid a good foundation for serological survey of DuCVs infection.