Functional Study On Bm64 Gene From Bombyx Mori Nucleopolyhedrovirus

Bombyx mori nucleopolyhedrovirus(Bm NPV) belongs to the genera ofNucleopolyhedrovirus in the family of Baculoviridae. Bm NPV is an importantpathgon to silkworm. Bm NPV infection caused serious losses to silkworm breedingevery year in China. Recent years, researches on Bm NPV gene function had becomehighlight in molecular biology of Bm NPV. But there are still a lot of unknownfunction genes.In this study, an unknown function bm64 gene of Bm NPV was studied. Accordingto whole genome sequence of Bm NPV T3 strain published on the NCBI(Gen BankNo. L33180.1), bm64 sequence was used to bioinformatics analysis. The homologywas 95% in bm64 gene between Ac MNPV and Bm NPV. Bm64 contained ahydrophobic domain(62-84 aa). p GEX-4T-3-bm64 plasmid was constructed andtransformed into Rosetta competent cell. Bm64 protein was expressed, affinitypurified, and immuned to the New Zealand rabbit to obtain polyclonal antibody.Bm64 protein expression was detected in Bm N cells infected by Bm NPV with theantibody we prepared. The indirect immunofluorescence assay showed that Bm64 protein located in the nucleus. To investigate the function of bm64 gene, Redhomologous recombination system and Bacmid were used. bm64 knockout bacmidwas constructed. In addition, bm64 gene rescued bacmid was constructed with bm64 promoter and ORF through Bac-to-Bac system. Meanwhile, egfp and ph genes wereinserted to different bacmid through Bac-to-Bac system to constructed three differentbacmids(Bacmid-egfp-ph, Bacmid-⊿bm64-egfp-ph, Bacmid-bm64rep-egfp-ph).Thethree bacmids were transfected into Bm N cells respectively. No Bm64 protein wasdetected by Western blot in bm64-knockout bacmid transfected cells. Cytopathogeniceffect of cells transfected with bm64 knockout bacmid significantly delayed. TCID50 of cells transfected with bm64 knockout bacmid reduced compared to the wild-typebacmid. The bm64-rescued recombinant virus showed the same phenotype towild-type virus. These results suggested that bm64 played an important role in virusreplication, but it play an important role in the infection of virus replication.Real-timePCR analysis showed transcription phage of bm64 gene was close to vp39 which wasa late gene. It suggested the bm64 gene might be a late gene. Viral DNA replicationand the transcription levels of lef-3, vp39 and p10 were diminished due to bm64 genedeletion.In conclusion, bm64 gene was a late gene, which localized in nucleus during virusinfection. This gene played an important role in virus replication.