Study On The Function Of Open Reading Frame(ORF)55 From Agrotis Segetum Granulovirus (AsGV)

ENHANCIN is a metalloproteinase, mainly encoded by Baculovirus. It has been shown to facilitates Nucleopolyhedrovirus(NPV),Bacillus thuringiensis (Bt) and other microbial pesticides infections in lepidopterans,it has a strong potential for application.In this study, enhancin-like gene and its truncated N-terminal sequence of 1020bp and 2679bp were obtained from the AsGV genome by PCR amplification.Characteristic of enhancin-like gene was then analysed;Three genes were cloned into expression vector pET-32a(+)or pET-28a(+),recombinant expression vectors then transformed into E. coli (BL21 (DE3)), fusion proteins were induced with IPTG and detected by SDS-PAGE, Western Blot and affinity chromatography;N-terminal sequence of 1020bp prokaryotic expression products(N-1020) were purified,separated and injected into rabbit to prepare polyclonal antibody; To study the synergistic infected function of three prokaryotic expression products to baculovirus, fusion proteins were preliminary purified,then fed third-instar(6 days old ) H.ar larvae with different concentrations of HaNPV.Sequence analysis shows that the enhancin-like gene encoding product contains the conserved domain“HEXXH”, has no signal peptide,no a-helical transmembrane region, while it has a unique C-terminal proline rich region, these characteristics may be related to its mechanism of action; AsGV ENHANCIN specific antibodies were obtained;Bioassay experiments shows that, within the HaNPV concentration of 1.17×10~2PIBS/mL, 1.17×10~3PIBS/mL, 1.17×10~4PIBS/mL, the fusion proteins expressed by enhancin-like gene can increase larval mortality 16.25%,21.25%and12.5% respectively,it has significant synergistic infection.While the truncated N-1020 has no synergistic effect, the N-2679 protein has weak synergistic infection.As N-2679 protein discards C-terminal proline-rich region,suggesting that this C-terminal proline-rich region plays an important role in synergistic function. According to mammals study, proline-rich region acts as an intermediate material,we speculate that the fusion protein plays as an intermediate molecule to facilitate virus infection. This study provids an experimental basis for the function of ORF55 from Agrotis Segetum granulovirus (AsGV) , but also laies a foundation for the application and scale production of ENHANCIN, also enrichs the knowledge of the mechanisms ENHANCIN synergistic.