Study Of The ORF80 Gene Function In The HearNPV(Helicoverpa Armigera Nucleopolyhedrovirus)

Ha80 is highly conserved, homology with all completely sequenced baculovirus genomes, including lepidopteran NPVs, lepidopteran GVs, hymenopteran baculoviruses, and one dipteran baculovirus. This gene is one of the core baculovirus genes, yet its function remains unknown.We prepared the polyclonal antibodies of ORF80 and constructed a recombinant bacmid with ORF80 deleted.HzAM1 cells were transfected by wiled virus and recombinant bacmid to observe effect of gene for virus replication.We got the whole sequence of Ha80 from HearNPV Bacmid G4 by PCR and constructed the prokaryotic expression vector pMAL-c4E-Ha80.The recombinant plasmid was transformed into the Escherichi Coli TB1 to express the MBP-Ha80 fushion protein.Purificated MBP-Ha80 protein was injected into rabbits as antigen to prepare the polyclonal antibodies of ORF80.Finally,we got the anti-HearNPV ORF80 polyclonal antibodies with the titre of 2.56×10⁵ detected by ELISA.We use Red recombinant system to knock out ORF80 gene and got the HearNPV^△80 Bacmid. PCR and single digestion of genome to identify the HearNPV^△80 Bacmid. HzAM1 cells were transfected by HearNPV^△80 Bacmid and GFP was expressed in the cells. Westen blot shown that ORF80 was successfully deleted from HearNPV genome.The supernatant of HearNPV^△80 BV was collected and re-infected to HzAM1 cells . The fluorescence didn't appearance and cells were health after infections, indicating the ORF80 was essential for virion infection.