Characterization Of BmNPV Orf98 From Bombyx Mori Nucleopolyhedrovirus

Baculovirus is one of the largest groups in insect virus, it can be encoded protein types from 90 to 180..Bombyx mori nucleopolyhedrovirus’ s sequencing was completed in 1999.Most of the gene function has been reported,but there are still some genes function without annotation. The gene of Bombyx morinucleopolyhedrovirus orf98 was found in all Group I and the most of Group II genomes. It was not a highly conserved gene, as the deletion of this gene in Bm NPV(Bm98), it had no apparent effect on infectivity.The function of Bm98 was not report from now on. In order to study the Bm NPV orf98 gene’s specific function, we constructed a Bm NPV orf98 knockout Bacmid by λRed recombination, named Bm98-ko-Bacmid. Additionally, Bm98-re-Bacmid was constructed by Bac-to-Bac system. Bm N cells were infected with Three kinds of virus DNA(Wt Bacmid,Bm98-ko-Bacmid and Bm98-re-Bacmid), and the cells were respectively collected at12 h, 24 h, 48 h and 76 h, then determinated virus titer(TCID50) and Virus proliferation curve was drew. Bm N cells were infected with Three kinds of virus DNA(Wt Bacmid,Bm98-ko-Bacmid and Bm98-re-Bacmid), and cells were respectively collected at12 h,24 h,48 h and 72 h, total DNA was extracted using the eukaryotic DNA extraction kit. After Dpn I enzyme digestion for the night, fluorescence quantitative PCR method analyzed the effects on virus replication and transcription lacking of Bm NPV orf98 gene.Experimental results show that, after transfecting the DNA of Bm98-ko-Bacmid into Bm N cells, the knockout Bacmid is able to produce viral progeny, but the number of viral progeny has reduced, meanwhile the virus infection level of repair is recovered similarly to wild-type virus. Which indicate that, in incidence of Bm N cells would be delayed after the deletion of Bm NPV orf98. Assembly of virus is observed by ransmission electron microscope in 48 h phase. In addition, we also built Bm NPV orf98 prokaryotic expression vector and express the original nucleoprotein. The preparation of polyclonal antibodies,Western blot analysis. Results show that: The first, with longer duration of transfection, the protein expression of Bm NPV orf98 in Bm N cells also grow.At last, electron microscopy observation results that the deletion of Bm NPV orf98 gene, The packaging of virus particlesand assembly is inhibited. However, the wild type and type of supplement virus have produced a large number of virus packaged by capsule membrane, and the deletion of Bm NPV orf98 gene only produces a litte free virus. The results indicated that, The Bm NPV orf98 is nonessential for viral DNA replication, because of the deletion of Bm NPV orf98 gene, the transcriptional level of early gene lef3, late gene vp39 and very late gene p10 have been decreased obviously because of the lack of Bm NPV orf98 gene. All the results show that, Bm NPV orf98 gene is non-essential for virus replication, but the lack of the gene lead to the transcription level of early gene lef3, late gene vp39 and very late gene p10 have declined obviously. At the same time, the total protein encoded by Bm NPV orf98 genes increase with the extension of time.