Studies On Preparation Of Polyclonal Antibody And Construction Of Gene Deleted Bacmids For HearNPV Orf83 And Orf86

The function of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ORF83 and ORF86 genes is uncertain thus far. We prepared the polyclonal antibodies of ORF83 and ORF86, and constructed the gene deleted HearNPV bacmids for ORF83 and ORF86. The results were reported as following:HearNPV ORF83 has 498 bp encoding a protein with approximately 165 amino acids and a predicted molecular weight of 18 kDa, whose function is not yet clearly understood. We amplified the coding region of ORF83 from the HearNPV G4 genome by PCR and then the recombinant prokaryotic expression vector pET-32a was constructed. The recombinant plasmid was transformed into Escherichi Coli BL21(DE3) to express the His fusion protein in the bacteria. After purification, the fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. Detected by ELISA, the titer of the anti-HearNPV ORF83 polyclonal antibodies was 1:2.56×105.HearNPV ORF86 is a protein of HearNPV with a molecular mass of 36 kD, whose function have not been reported thus far. We amplified the coding region of ORF86 from the HearNPV G4 genome by PCR and then the recombinant prokaryotic expression vector pGEX-4T-2 was constructed. The recombinant plasmid was transformed into E.coli BL21 to express the GST fusion protein in the bacteria. After purification, the fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. Detected by ELISA, the titer of the anti-HearNPV ORF86 polyclonal antibodies was 1:5.12×105. The specificity of the anti-ORF86 antibody was successfully proved by western blotting on HZAM1 transfected by HearNPV. The successful preparation of polyclonal antibodies against ORF86 would facilitate the detection and related research of ORF86The homologous recombination fragment was PCR-amplified from PKSE plasmid, which have Egfp and CmR gene. The linear fragment was then transformed into E. coli Hz8/pKD46 electrocompetent cells, which have HearNPV bacmid and Red helper plasmid pKD46. The gene deleted HearNPV bacmids for ORF83 and ORF86 was confirmed by PCR and restriction enzyme analysis. The bacmids can be used to further study the function of gene ORF83 and ORF86 in the process of viral replication, transfecting cells and infecting insects.