Expression of channel catfish virus ORF6 gene in insect cells

Channel catfish virus(CCV) is a pathogen of channel catfish.This virus can cause severe hemorrhagic disease in young channel catfish and cause major losses to fish farmers.As no efficacious treatment exists,a vaccine against CCV is essential for prevention and control of this disease.Recently,the baculovirus was described as an attractive gene delivery vehicle and a potential vector for vaccine development.In order to express the membrane protein of the CCV ORF6 gene in insect cells,ORF6 gene was subcloned into baculovirus transfer vector(pFastBac^TM-1).The recombinant plasmid pFast-ORF6 was identified by restriction enzyme digestion and gene sequencing.DH10Bac containing baculovirus shutter vector(bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF6.The recombinant bacmid(rBacmid-ORF6) was identified by blue/white selection and PCR analysis.Then the rBacmid-ORF6 was transfected into sf9 cells with Cellfectin Reagent and recombinant baculovirus was obtained in sf9 cells,named AC-ORF6.AC-ORF6 was multiple-nucleopolyhedrovirus which was observed by electron microscopy in the ultra thin section of infected sf9 cells.After AC-ORF6 infection,the recombinant CCV ORF6 protein was characterized by indirect immunofluorescence assay and Western-blotting.The results showed that ORF6 protein was expressed in sf9 cells and had a molecular mass of 16 ku.This study will provide useful basis for the subunit CCV vaccine development in the future.