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

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.