Characterization Of ORF67 And ORF101 From Bombyx Mori Nucleopolyhedrovirus

The family Baculoviridae is a highly selective pathogen in arthropods,mainly in insects of the order Lepidoptera.Recently,the baculovirus have been successfully developed as expression vector system,recombinant bio-insecticide,gene transfer vector and surface display vector,providing huge benefits to nature science development.From the known genes,some are involved in the compositon of BV or ODV,some are involved in the virus replication,transcription and structural assemblage, and several other genes whose function are unknown.In this study,two baculovirus genes,Bombyx mori nucleopolyhedrovirus(BmNPV)ORF67,ORF101 were characterized.The gene transcription,gene expression,sub-cellular location,virus structural location and gene knock-out were adopted to characterize these four genes. The object of this study is to enhance our understanding of baculovirus molecular biology.The results were as following:BmORF67 is located between nt61188 and nt61892 of the genome,containing 705 bp and coding 234 aa with predicted molecular weight 27 kDa.Sequence analysis indicated that BmORF67 is a conserved gene,whose homologues can be found in all the genome of baculovirus.BmORF101 gene,between nt 95620 and nt 96354 of the genome,contains 735 bp and codes 244 aa with predicted molecular weight 28.1 kDa. The homologues of BmORF101 were found in 11 genomes of baculovirus.Two primers were designed as the BmORF67 gene sequence and used for PCR amplification.PCR products were cloned into pET30a(+)vector and BmORF67 were expressed as fusion protein.The purified fusion proteins were injected into New Zealand white rabbits to harvest polyclonal antibodies.Western blot analysis of extracts from BmNPV-infected BmN cells revealed a specific polypeptide with an apparent size of 27kDa when using the BmORF67 antiserum.RT-PCR results showed that BmORF67 genes transcribed in the infected cells from 18h post infection(p.i.)to 72h p.i.Western blot analysis showed that BmORF67 was detected in infected cells from 24 to 72h p.i.which further suggesting that BmORF67 is a late gene.BmORF67 was not detected either in budded viruses or occlusion-derived virus.The sub-cellular localization of the BmORF67 proteins was investigated by immmunofluorescence.The fluorescence was concentrated on the cytoplasm and no fluorescence was detected in the nucleoplasm.Using His pull-down, co-immunoprecipitation and peptide mass fingerprinting with MALDI-TOF,it was found that BmORF67 interacted with host protein actin A3.BmORF101 deletion bacmid was generated by Red-recombination in E.coli DH10B.An BmORF101 gene fragment was knocked out from the BmNPV bacmid and a Cm gene was inserted into the same site.The gfp and polh genes were transposed into the recombinant bacmid that was used to infect BmN cells.Green flucrescence can be seen in 24h p.i.BmN cells transfected by recombinant bacmid and the cells with green flucrescence scattered equally in the vision and remained few till 96h p.i.just as the same amount of 24 h p.i.cells.The level of BV production was detected to be zero in recombinant bacmid transfecting cells.Thus,the BmORF101-recovered virus was constructed.The amount of infected cells with fluorescence continuously increased after the cells transfected with BmORF101-recovered DNA,which demonstrated that the virus rescued the disability successfully.This phenomenon was confirmed by the second generation infection.Thus it is concluded that the BmORF101 gene is an essential gene for BV production.RT-PCR results showed that BmORF101 genes transcribed in the infected cells from 24h post infection(p.i.)to 72h p.i.,suggesting it was a late gene.BmORF101 fused with GFP was used to visualize its sub-cellular location.First,BmORF101 was located in the cytoplasm and then some fluorescence was transfected to the nucleoplasm.