鸡传染性法氏囊病病毒Gt株VP5基因缺失株感染性克隆的构建

Cloning of infectious bursal disease virus （Gt strain） lack of VP5 gene

摘要:

传染性法氏囊病病毒 (IBDV)是双链，双节段RNA病毒，其基因组由A、B两个节段组成，编码结构蛋白VP1-VP4和非结构蛋白VP5。【目的】利用反向遗传操作构建拯救VP5基因缺失重组IBDV。【方法】利用体外定点突变技术，缺失IBDV Gt株VP5基因，通过多重PCR在基因组两端分别引入锤头状核酶序列(HamRz)和丁肝病毒核酶序列(HdvRz)。将带有核酶序列的IBDV基因组插入载体pCAGG的b肌动蛋白启动子下游，构建了IBDV感染性克隆pCAGGmGtA △VP5HRT，将该感染性克隆与pCAGGmGtBHRT共转染DFⅠ细胞。【结果】RT-PCR和间接免疫荧光均显示获得重组病毒，将其命名为rmGtA △VP5。IBDV VP5基因缺失感染性克隆的成功构建为从分子水平上深入研究vp5基因功能奠定了基础。

Abstract:

Infectious bursal disease virus （IBDV) belongs to genus Avibirnavirus of the family Birnaviridae. The genome of IBDV consists of two segments of double-strand RNA, which encode four structural protein VP1-VP4 and one non-structural protein VP5. [Objective]To study the function of VP5 of IBDV, the recombinant virus, lack of VP5 gene, was constructed and rescused by reverse genetic technique. [Methods] We deleted the VP5 gene （on segment A) of IBDV Gt strain by silence the start codon （ATG-ATC) using site-directed mutagenesis. The full length cDNA of segment A was flanked by hammerhead ribozyme and hepatitis delta virus ribozyme, which was introduced into an eukaryotic expression vector PCAGGS, under the strong chicken b actin promoter. The recombinant plasmid was named as pCAGGmGtA △VP5HRT. Co-transfection was carried with PCAGGmGtAdVP5HRT and PCAGGmGtBHRT in DF-I cells. [Results] Recombinant virus was successfully rescused, which was verified by RT-PCR and indirect immnuofluorescence assay. The rescusd virus could be a very helpful platform for further study of VP5 biological function.