The Initial Research Of Cellular Location And Infection Related Effection Of Duck Enteritis Virus Gd Protein

Duck viral enteritis (DVE), also known as duck plague (DP), which is an acute, heat and haemorrhagic contagious viral disease in infected birds of the order Anseriformes(ducks, geese and swans). DEV was the etiological agent, which was assigned as member of family Herpesviridae, unclassified virus. The glycoprotein D is virulence protein, but promotes the process of penetration and cell-to-cell spread. gD proteins combined specifically with the TAP, which inhibits the antigen-presenting pathway of MHC I molecules. Therefore, function research of gD proteins has significance in further understanding the mechanism of viral infection.In this study, duck enteritis virus (DEV) C-KCE genomic DNA as template, us6ORF was amplified by PCR. Then detailed bioinformatics of gD protein was analyzed by software, including amino acid sequence and protein structure.47KDa recombinant protein was gained by E. coli expression system pET30a (+)(E. coli Rosetta). The purified protein was used to immunize rabbits to preparate the rabbit anti-gDw polyclonal antibody, serum titer was tested by agar diffusion and plaque reduction neutralization test. The antibody has good responsiveness for the DEV gD protein by western blot and indirect immunofluorescence assay analysis.The cell lysates of DEV infected has been real-time harvested, the extractiion of total RNA was used to analysis the gD gene transcription by RT-PCR. The us6gene transcription could be detected after2h of DEV infected, which in line with the characteristics of γ1gene. The rabbit anti DEV-gDw IgG has been used to characterize cellular localization of the proteins by indirect immunofluorescence. The specific green fluorescent appeared in the nuclear membrane and perinuclear vesicles as early as4h post infection. After12h post infection the newly synthesized gD widely presented in the cytoplasm, and after72h gD was concentrated in the cell membrane.Anti-gDw antibodies were added when DEV inoculation in duck embryo fibroblast culture, in order to detect the role of gD protein in adsorption, penetration, direct cell-to-cell spread (CTCS), as well as the impact of the virus replication in vitro. The results showed that the anti-gDw IgG adding experimental group compared with the control group, virus penetration rate and the formation of plaque size had significant differences, but the adsorption rate had barely difference. DEV gD protein may be involved in invasion and intercellular virus transmission process. Added anti-gDw IgG in vitro,the emergence of CPE was delayed but the characteristics of proliferation was barely diierent.