Primary Study Of The Characteristics Of Orf109 From Helicoverpa Armigera Single Nucleocapsid Nucleopolyhedrovirus

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is a kind of large doublestranded cycle DNA virus, great progress has been made in its molecular and functional research. Open reading frame 109(orf109) is a conserved gene, whose function is unknown. This paper reported the primary study results of orf109.Bioinformatic anaysis shows that orf109 is 357 bp long, which encodes a putative protein of 118 amino acids and its predicted molecular size is 13.6 kDa. The protein was named HA 109. Two early baculoviral transcription initation motif CAGT and TATAA was found at -235 and -238nt upstream of the putative translational start site separately. Ha109 is unique to an unknown gene of HaSNPV G4 line features, because of its nuclear export signal(NES), suggesting that this gene is likely to play a specific role,such as matter and energy in the host cell nuclear transport and signal transduction; The results of BLAST found that it only has 99% homology with the HzSNPV orf112, only a few nucleotide differences, but it does not affect the encoded protein sequence, so the nucleic acid sequence differences reflect the different evolutionary; the orf109 is likely to be the new genes of recombination and rearrangement in the virus evolution, but the exact mechanism is unclear. So Ha109 gene function is important to clarify the uniqueness and evolutionary history of the gene.HA109 was expressed in E.coli BL21 with GST-fusion expression vector pGEX-KG. After IPTG induction, the E.coli BL21 containing recombinant plasmids expressed a considerable fusion protein of GST-HA109 in form of inclusion body. The molecular weight of the fusion protein is 38.5 KDa, which is in agreement with the anticipation. The fusion protein GST-HA109 was purified by SDS-PAGE, and used to generate antibodies in New zealand white rabbit. Western blot analysis using the multiclonal antibodies with 1:8000 diluted showed a specific reaction to GST-HA109 fusion protein expressed in E.coli BL21. The preparation of the multiclonal antibodies made it possible to analyze the localization of HA109 in virion and its expression phase in infected insect cells. We also try to find out soluble expression condition of GST-HA109, however, we found that GST-HA109 mainly expressed in form of inclusion body, and very little soluble protein was detected.In order to know the function of orf109, we constructed the recombinant HaSNPV with orf109 deletion in E. coli BW25113. In this part, we cloned the upstream homologous arm and homologous downstream arm of orf109 into the two ends of Phsp7o-egfp-SV4o-Cmr separately in the plamid of pKS-egfp-Cmr, and get the linear fragment of ha109UP-Phsp70-egfp-Cmr-ha109DO by restriction enzyme digestion, which was used to homologous recombination. The helper plasmid pKD46 encodesγphage Red-ET recombination enzyme system, and the recombination will occur between bacterial artificial chromosome HaBacHz8 and linear fragment ha109UP-Phsp7o-egfp-Cmr-ha109DO with the help of pKD46. After PCR and restriction enzyme digestion detection, we got the recombinant HaBacA109.For system analysis the function of orf109 in the replication cycle of HaSNPV, we construct four different genotype recombinant bacmids:orf109 knockout bacmid, HaBacKO; orf109 single repair bacmid, HaBacSingleRep; orf109 double repair bacmid, HaBacDualRep; wildtype control bacmid, HaBacWt. All genotype bacmids of above were accompanied with egfp gene and polyhedrin gene derived from HaSNPV, and this was accomplished through Bac-to-Bac system. The three genotype bacmids made it possible to analyze the impact of orf109 deletion on the genome replication, the titre of budded virus(BV) and oclusion-derived virus (ODV) assembly etc., then we could determine its exact function.