Characterization Of Bm94 From Bombyx Mori Nucleocapsid Nucleopolyhedrovirus

Baculoviruses are one of the largest group of insect viruses and the viruses were discovered at the earliest time. Baculoviruses have been widely studied and been employed in multiple research areas and in industry. Recent years, baculoviruses have been wildly used as efficient eukaryotic expression vector, insecticides for biological protection of forest and agricultural pests surface display vector, and gene therapy vector.It is generally believed that core genes play an important role in RNA transcription, DNA replication, while the Species-specific genes or Genus-specific genes may specificly involve in the interactions between hosts and virus. In this article, Bombyx mori nucleopolyhedrovirus(BmNPV)Bm94, one of the non-core gene was studied. Study of Bm94 will shed light on its role in our next work. The main results are shown as below:BmNPV orf94 is located between 90,089 nt and 91,363 nt in BmNPV genome, which encodes 424 amino acid residues with predicted molecular weight of 49.5 kDa. Bm94 belongs to the specific genes of group I lepidopteral baculovirus, which is also a Species-specific gene. Blast search results revealed that Bm94 homologues exist in 10 baculovirus NPVs with identities ranging from 95% to 37%, Sequence comparison of the amino acid (aa) sequences shows that Bm94 has its highest identity (95%) with AcMNPV ORF114 and its lowest identity (37%) with OpMNFV ORF114.Primers were designed to amply the Bm94 by PCR and BM94 protein was expressed in E.coli as a fusion protein using the pET-30a vector. The result showed that BM94 could be efficiently expressed in E.coli at a size of about 49.5 kDa. Western blotting and mass spectrometry analysis proved that the recombination protein expressed in E.coli BL21 is recombinant protein of His- BM94.RT-PCR analysis of Bm94 transcripts showed that the transcript of Bm94 gene can be detected from 12h p.i to 72h p.i. But the immunodetection of Bm94 protein in BmNPV-infected cells was not detected until 24h p.i. To investigate the subcellular localization of BM94, we peformed the immunofluorescence analysis. The staining results showed that fluorescence was observed in the BmN cell cytoplasm at 24 h p.i., 48 h p.i., and 72 h p.i., it shows that the BM94 protein only appears at cell cytoplasm. In order to investigate its role, Bm94 was rapidly disrupted in E.coli by Red recombinant system.