Cloning, Expression And Partial Biological Functions Of Duck Enteritis Virus GC Gene

Duck viral enteritis (DVE), also known as duck plague, is an acute, heat, haemorrhagic and lethal disease. The disease is caused by duck enteritis virus (DEV), which was epidemic in many countries and regions and caused economic losses. DEV is a member of of family Herpesviridae. According to the report of Eighth International Committee on Taxonomy of Virus (ICTV), DEV was assigned as member of family Herpesviridae, unclassified virus. Glycoprotein C(gC)is a secondary envelope protein of herpesvirus and plays an important role in virus attachment, release and virulence. Furthermore, it could stimulate body produce neutralization antibody. Therefore, the research on gC have significance not only the function and characterization in the molecular biology of the virus but the clinical diagnosis, prevention, genetically engineering vaccine.In present study, using genomic DNA of duck enteritis virus as template, TAIL-PCRs method were performed. The DEV C-KCE gC and flank sequences was obtained, including partial UL43 open reading frame (ORF), UL44(gC) and noncoding region between gC and UL45. And they were subcloned into pMD18-Tvecter. The recombinant plasmid pMD18-T-UL45-44 and pMD18-T- UL44-43 were constrcted and then sequence analysis revealed a 1296bp ORF encoding a 431 amino acid(aa) polypeptide homologous to herpesvirus gC proteins,including 2 potential promoters and poly(A) signals, a 600bp partial ORF encoding a 200aa polypeptide homologous to herpesvirus UL43 proteins. The part of 1-22, 1-398, 399-421 and 422-431 of gC were predicted as the signal peptide, extracellular, transmembrane or cytoplasmic domain of gC, respectively. In addition, four potential N-glycosylation sites were found of the DEV gC. Sequence comparison of gC(UL44) with gC of 12 other reference herpesvirus strains showed that DEV C-KCE was homologous to Mardivirus of alphaherpesviruses. A clustered alignment of amino acid sequences showed that the gC(UL44) of DEV C-KCE had three conserved regions with alphaherpesviruses. Phylogenetic analysis of the gC gene of DEV showed that DEV was more closely related to avian herpesviruses than to other herpesviruses.The pET-30a(+) (E. coli Rosetta) prokaryotic expression system was used to express the extracellular part of gC without the signal sequence. The results showed that the high expression level of it. The pET 30a(+) (E. coli Rosetta) system expressed insoluble fusion protein His-gC was 30% of total bacterial protein and the Mr was about 49kDa. Western blot analysis proved that the fusion protein(gC trcunted) has specific antigenicity. After purification, the fusion protein was used to immunize the rabbits for preparing antibodies. And it proved that the antibodies have high specificity.Using DEV grew in CEF as antigen, the sera of rabbit anti-gC as detecting antibodies, indirect immunofluorescence was performed. The results show that glycoprotein C lied in perinuclear region and membrane of the CEF cells.Using rabbit anti-gC antibodies as neutralization antibody, plague reduced neutralization assay was performed. The results show that the antibody can neutralize DEV. According to the result, the gC has neutralization activity.Using recombination gC protein as stimulus, Duck's Proliferation of Lymphocyte Experiment was proformed, the result showed that glycoprotein C can stimulate T cells. Detecting the level of IFN-γof cell supernatant by duck IFN-γELISA kit(USCN,USA), the results showed that gC can stimulus lymphocyte produce IFN-γ. Therefore, gC can stimulus cytoimmunity of body.