摘要
鸽圆环病毒(Pigeon Circovirus,PiCV)是近年来发现的圆环病毒科的一个新成员。与其他圆环病毒一样,鸽圆环病毒常潜伏感染,但往往侵染淋巴细胞等增殖速度快的细胞,引起免疫力下降,导致动物对多种条件性病原二次感染的易感性增加。
根据已发表的鸽圆环病毒序列,设计了一对检测引物,对浙江某鸽养殖场患病鸽进行PCR扩增,获得与预期629 bp大小相符的DNA片段,经测序比对确认为PiCV特异序列,再设计三对引物扩增PiCV基因组的片段,经测序、四片段拼接后获得PiCV全长基因组序列。
基因组序列分析表明,浙江株PiCV全长2039bp,具有圆环病毒共同的与病毒复制相关的茎环结构和Rep蛋白保守基序等特征,PiCV-zj1和PiCV-zj2与已发表的3株PiCV全基因组序列的同源性分别在88.1%—88.7%及88%—88.3%之间,而PiCV-zj1、PiCV-zj2之间的同源性为94.2%。应用PHYLIP程序作进化树分析表明PiCV-zj1及PiCV-zj2与德国株、英国株可分成2个组,其中浙江两株成一分支,其余3株成另一分支。
应用设计的删除核定位信号外壳蛋白基因(△Cap)的引物,扩增获得680bp的△Cap基因,克隆于pMD18-T载体,进行测序；将△Cap基因亚克隆到原核表达载体pET-28a进行融合表达,SDS-PAGE电泳检测发现,△Cap融合蛋白经IPTG诱导后在大肠杆菌中以包涵体形式表达,目的蛋白表达量占菌体总蛋白的22.1%。PiCVΔCap的高效表达为PiCV诊断试剂盒的研制和血清流行病学调查奠定了基础。
Pigeon circovirus was a new member of circoviridae recongzied last few years. Like other circoviruses, it often causes subclinical disease rather than clinical disease.However, circoviurses invade lymphoid tissue and lead to immuno-supression, growth retardation and an increased probability of secondary infections.
A pair of degenerated primers, which amplified a fragment of 629bp in length, was designed and synthesized, based on the published goose and other circovirus sequences. The specific PCR product was amplified from the pigeon sample of Zhejiang Province. Three more pair of primers which amplied three DNA fragments,then the four DNA fragments covered the complete genome of PiCV.
We reported two complete nucleotide sequences of pigeon circovirus isolated from Zhejiang province of China. The two strains of PiCV, named PiCV-zj1 and PiCV-zj2, were isolated and cloned by using PCR method. Sequences analysis of PiCV-zj1 and PiCV-zj2 showed that both of them were 2039 bp in length and coded five ORFs, ORF V1, C1, C2, C3 and C4. PHYLIP analysis of the nucleotide sequence of PiCVs isolated from Zhejiang together with published sequences showed 88%-88.7% identical residues. Phylogenic analysis demonstrated that PiCVs comprised two distinct groups. GroupⅠcontained two Zhejiang isolates, while groupⅡcontained Germany isolates and Northern Ireland of U.K. isolates.
A pair of primers which amplified△Cap gene, a Cap gene that the coding region of nuclear localization signal was excluded,680bp in length was designed. The△Cap gene was amplified, cloned into pMD18-T and sequenced. And the gene was then subcloned into the pET-28a. SDS-PAGE analysis showed that the recombinant protein was expressed, which existed as inclusion body and accumulated up to 22.1% of the total bacteria protein. The way could have value as a method for further study of PiCV.
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