Partial Biological Functions Of Duck Enteritis Virus GE And Construction Of GE Gene Transfer Vector

Duck viral enteritis (DVE), also known as duck plague (DP), its pathogen is duck enteritis virus (DEV), which is acute, heat and haemorrhagic contagious viral disease in infected birds of the order Anseriformes (ducks, geese and swans). DEV is characterized by epidemic broadly, spread rapidly with high mortality and morbidity. Glycoprotein E (gE), the product of the us8 gene is one of the main envelope glycoprotein of herpesviridae virus. gE is unnecessary for herpesvirus replication and plays an important role in virus cell to cell spread and virulence. Furthermore, it could stimulate body produce antibody and cell-mediated immune responses, and has been shown to be a candidate antigen for recombinant, subunit vaccines, serology diagnostic assays. Therefore, the research on gE have significance not only the function and characterization in the molecular biology of the virus but the clinical diagnosis, prevention, genetically engineering vaccine.There are very limited studies on gE gene and gE protein of DEV. In present study, the DEV gE gene was obtained by PCR with genomic DNA of duck enteritis virus C-KCE strain as template. The homology of gE gene and gE protein with other herpesvirus, the structure and antigenicity of gE protein were analysised by bioinformatics. On this basis, the pET-30a(+)(E.coli Rosetta) prokaryotic expression system was used to express the gE mature protein (pET-gE), extracellular part of gE mature protein (pET-gEBW) and cytoplasmic part of gE mature protein (pET-gEBN). The recombinant protein of pET-gE, pET-gEBW and pET-gEBN about 60 KDa, 50 KDa and 20 KDa were produced after induction.Western blot analysis proved that fusion proteins can react with positive sera from duck, and shows a good antigenicity.The pET-gEBW fusion proteins was purificated by gel extraction method and used to immunize the rabbits for preparing polyclonal antibodies. And the analysis by western blot and indirect immunofluorescence assay (IFA) proved that the antibodies have high specificity, could react with the recombinant protein and native protein.The rabbit anti DEV-gE antibody has been used to characterize cellular localization of the proteins. The results showed that glycoprotein E lied in perinuclear region and membrane of the CEF cells. Plague reduced neutralization assay was performed with using rabbit anti DEV-gE antibody as neutralization antibody,.The results showed that the antibody can neutralize DEV. According to the result, the gE has neutralization activity. The gE function which contain attachment, penetration and direct cell-to-cell spread (CTCS) were analyzed by adding the gE antibody to the culture medium of the primary CEF cell monolayers. The treatment with rabbit anti DEV-gE antibody had no obvious affection to the virus penetration. The relative penetration efficiencies of the DEV C-KCE strain and anti-gE antibody treated DEV C-KCE strain were 53.24% and 51.39% respectively. The attachment efficiencies of the DEV C-KCE strain and anti-gE antibody treated DEV C-KCE strain were 63.50% and 45.31 % respectively. Attachment efficiencies were reduced significantly (p<0.05) treated with gE antibody showed that gE was involved in virus attachment. The gE antibody reduced the virus plaque size (72.8% percent of the control), which indicated that gE played a role in cell-to-cell spread.In addition, the gE gene (including gI and its flanking sequences) was amplified by polymerase chain reaction (PCR) with extracted total DNA from DEV C-KCE strain as template and cloned into vector pGEM-T. Then the gE gene was subcloned into pUC19 vector which digested by EcoRâ… and SaIâ… to construct a recombinant vector pUC-gE. At last the EGFP expression cassette was inserted into BamHâ… sites of the pUC-gE vector to obtain the DEV transfer vector pUC-gE-EGFP. The transfer vector pUC-gE-EGFP was transfected into DEF cell, then the green fluorescence could be seen, the results showed that EGFP gene could be expressed correctly. It's useful for developing the recombinant DEV expressing foreign gene(s).