Antigen Analysis Of Duck Tembusu Virus Envelope Protein

The newly emerged duck Tembusu virus disease characterized by rapid-egg-drop had resulted in a massive economic loss to the duck industry in China. The causative agent of the disease was duck Tembusu virus (DTMUV), which belongs to the genus of flavivirus in flaviviridea. To date, the pathogenesis of DTMUV is largely unknown and there is no effective control measure for prevention of the disease. The envelope glycoprotein, a major structural antigen, is associated with viral attachment, fusion, cellular tropism, viral virulence, and induction of protective immune response. In this study, we investigated the immunogenicity of the E protein domain III and identify the linear epitope of E protein, which will lay a good foundation for the vaccine development.In this study, we developed a micro-neutralization assay which employed a monoclonal antibody reactive with E protein of DTMUV. The serum-virus mixtures were added to the96-well plates that has grown with a monolayer DF-1cells. After incubated24h, the neutralizing titers were assessed by using a indirect fluorescent assay (IFA). In this study, the newly developed neutralization assay was highly specific. Compared to the neutralizing test in chicken embrynated eggs, this new assay have a lot of advantages, such much more stable, sensitive and time-saving, high-throughput and easy to operate. Compared to the micro-neutralization test based on inhibition of cytopathic effect, this new assay was much more sensitive and time-saving.30clinical serum samples detected by the method were further confirmed that the method has good usability.In order to understand whether prokaryotic expression EDIIII of DTMUV can induce neutralizing antibodies. The coding sequence of D? was optimized (optiDIII) according to the bias codon usages of E.coli and then cloned into the multiple cloning site of pCold-TF vector to construct the recombinant plasmid pCold-TF-optiDIII. The expression of recombinant EDIII was confirmed in SDS-PAGE. The reaction between EDIII and anti-DTMUV serum was confirmed in Western Blot as well. After immunized BALB/c mice three times with EDIII, humoral immune responses specific for DTMUV were also demonstrated in mouse serum samples using ELISA, IFA. In addition, the neutralizing activity of immunized serum was at dilutions up to1:80in neutralization assay. These findings have indicated that the recombinant D? can be used for development of diagnostic reagents and subunit vaccines of DTMUV.In order to fully identify the DTMUV E protein linear epitopes and mapping of the E protein epitope, we designed and synthesized41partially overlapping peptides which covering the full length of the E protein amino acids in this study. The synthetic peptide coupled to a carrier protein KLH and immunized BALB/c mice, using indirect ELISA and indirect immunofluorescence assay detection of immune serum reactivity with DTMUV. Finally, five linear epitopes were identified, namely:"LEGGSCVTITAKDKPTIDVK44,97VDRGWGNG104,229SAGTWQNK236,349MTPVGRLI356and373LVGSGKGQIRYQWHRSGSTI392. After BLAST in the Immune Epitope Database (IEDB), apart from229-236aa, the remaining epitope sequences have high similarity with other epitopes which have been identified in other flaviviruses. However, the neutralization assay was proved that all these epitopes can't induce neutralizing antibody against DTMUV. These results provided important basis for developing epitope-based diagnostic methods.