The Eukaryotic Expression,Subcellular Localization,Polyclonal Antibody Preparation And Molecular Characterization Analysis Of Duck Plague Virus UL22 Gene

In this paper, we study the eukaryotic expression, subcellular localization, polyclonal antibody preparation and molecular characterization analysis of duck plague virus UL22 gene. We have obtained the following results:1.Bioinformatics analysis of DPV UL22 geneThe nucleotide sequence of the UL22 gene, glycoprotein H encoded by UL22 gene and the homologous with other herpes viruses were analyzed through bioinformatic analysis tools. The results indicated that the UL22 gene was 2505bp and translated into a protein comprising 834 amino acids with a putative molecular mass of 92.5433kDa. There were a signal peptide, two transmembrane regions,8 N-glycosylation sites and 52 potential phosphorylation sites. There were three disulfide bond formation between gH molecules and many antigenic sites. Homology analysis showed that gH was very conservative in the herpes virus and had close evolutionary relationship with the MeHV-1, GaHV-2 and GaHV-3 which were belong to Mardivirus genus.2. Prokaryotic expression and antibody preparation of DPV gHt geneAccording to the bioinformatic prediction, we removed the signal peptide and the transmembrane region of gH to obtain a truncatedform gHt. Then we constructed prokaryotic expression plasmid pET32c(+)-gHt. The plasmid pET32c(+)-gHt was transformed into the express host bacterium BL21 and induced expression at 37℃, 0.2mmol/L IPTG and 10h conditions. The protein was mainly present in the inclusion bodies. The titer of hyperimmune serum which was prepared by using purified protein gHt to immune rabbit reached 1:32, Western blot have confirmed gHt protein can be recognized by sera of rabbit anti-DPV.3. Intracellular localization of DPV glycoprotein HBy indirect immunofluorescence assay to detect DPV gH expression and localization in the infected cells, experimental results showed that gH was found mainly in the cytoplasm, consistent with bioinformatic prediction results. gH began to express after the viral infection 6h. The expression amount was maximum in 30-43h.4. Construction and expression analysis of a. eukaryotic recombinant plasmid encoding duck plague virus UL22 geneThe constructed T cloning plasmid of pMD20T-gH and pMD19T-gHt were dealed with double digestion, and then connected with the eukaryotic expression vector pCMV-HA, building. recombinant eukaryotic expression plasmid pCMV-HA-gH and pCMV-HA-gHt.’Recombinant plasmid was transfected HEK293 cell. by liposome LipofectamineTM2000, and then analyzed the expression by RT-PCR, indirect immunofluorescence test. The results showed that the recombinant plasmid pCMV-HA-gH and pCMV-HA-gHt had high levels of expression and located mainly in the cytoplasm in HEK293 cell. All these results provided a reference to establish a stable expression cell lines of DPV gH.