Expression And Intracellular Localization Of Duck Plaque Virus Strain CHv VP19c Protein And Preliminary Study On Inhibition Of Virus Replication By RNA Interference

There are very limited studies on VP19c protein of DPV. In present study, bioinformatics analysis, clone and prokaryotic expression, protein purification and preparing polyclonal antibodies, expression phase and intracellular localization of the DPV UL38 gene (accession no. EU071041) which was registered to NCBI Gene Bank by our lab and the encoding protein were studied. The results follow as:1. Bioinformatics analysis of UL38 gene The structure, motif, antigenicity and rare codon of UL38 gene and the encoding protein were analysed by bioinformatics. The results showed its encoding protein (VP19c) had a Herpes_ VP19c conserved domain. Moreover, the nucleic acid and amino acid sequence of DPV UL38 had higher homology with UL38 homologous protein of Alphaherpesvirus than others. DPV VP19c contained no signal peptide and transmembrane domain and can target to nucleus of cell.It had abundant epitope and most of them located in N terminus of the polypeptide. In addition, UL38 gene had no more contiguous rare codon.2. Cloning, prokaryotic expression, preparation of polyclonal antibody of UL38 gene On this basis, the gene was cloned and the pET-32a(+) prokaryotic expression system was used to express the protein encoded by the ORF and part ORF. The recombinant proteins about 66 kDa and 45 kDa were produced after induction.Western blot analysis proved that fusion proteins could react with anti-DPV polyclonal antibodies, and ELISA analysis showed the similar antigenicity of the two proteins.The fusion proteins were purified and used to immunize the rabbits for preparing polyclonal antibodies. The antibodies titer was 1:8. And the analysis by western blot proved that the antibodies could react with the native protein of DPV.3. Expression phase and intracellular localization of VP19c protein Western blot analysis proved that VP19c protein could be detected at the time of 8 h post infection. The expression phase of VP19c protein was similar with the time courses expression of classic late protein from herpesviruses. The polyclonal antibody has been also used to characterize intracellular localization of the protein.The result showed that VP19c protein was expressed diffusely throughout the cytoplasm of cells at 8 h.p.i. At late times (30h) following infection, the VP19c protein localized in very fine punctate forms dispersed throughout the nucleus of infected cells. To study deeply the cellular localization of VP19c protein, a fusion expression plasmids,containing EGFP gene and UL38 gene, were constructed. DEF cells were examined by fluorescence microscope after being transfected with the fusion expression plasmids. The result showed that VP19c protein was located in cell nucleus, which suggested it contain a NLS.4. Inhibition of DPV replication by RNA interference targeted to UL38 gene There are very limited studies on inhibition of DPV replication by RNAi vector. This researeh in view of DPV strsin CHv UL38 gene, known sequences of waterfowl, strategy of designing shRNA and selecting siRNA target sequence,3 siRNA target sequence were selected.The three shRNA vectors were constructed by Shanghai JiKai. DEF cells were examined by fluorescence microscope after being con-transfected with the fusion expression plasmids and shRNA vectors. The result showed that a shRNA vectors have good inhibition ability.Then, the CPE was examined after being transfected with shRNA vector and DPV infection. The result showed that the shRNA vector could inhibit the replication of DPV.