Genome Sequence Analysis Of Duck Hepatitis Virus Type 1 JX Strain And Prokaryotic Expression Of VP0 And VP1 Gene

DHV-1 is now worldwide in distribution and is the one of most economic importance to all duck-growing farms because of the high mortality. To date, no research of protein function for DHV-1 has been reported, only the genomic structure and proteidic organization predicted, which greatly hindered the relevant research. In this study, the genome sequence of DHV-1 JX strain was determined and analyzed, and the structure protein VP0 and VP1 gene were expressed in prokaryotic expression system, which is important for the research of molecular epidemiology of DHV-1 and vaccines for DHV-1. There are three parts in the thesis.In the first part, after the sequences alignment of three DHV-1 strains which published in GenBank, nine overlapping pairs of primers were designed based on conserved motifs. Genome sequence of DHV-1 JX strain was amplified by reverse transcription polymerase chain reaction (RT-PCR). The products of RT-PCR were cloned into the pMD18-T vector, and then transformed to competent E.coli DH5αcells. Positive clones were screened and determined by RCR. The sequences of positive clones were determined by automated sequencer. The nucleotide sequence of DHV-1 JX strain genome has been submitted to the GenBank database and assigned accession no. EF093502. As a result, the genome of DHV-1 JX strain is 7582 nt, and is composed of 5' untranslated region (UTR), 3' untranslated region (UTR) and a single, large open reading frame(ORF) encoding a polyprotein of 2249 aa. The genomic structure of DHV-1 JX strain is organized as: 5'UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3'UTR.In the second part, the genome sequence of DHV-1 JX strain was compared with those of other DHV-1 strains published in GenBank. Their genetic characteristic and evolutionary relationship were analyzed by molecular biological software DNA Star. Sequence homology analysis showed that the DHV-1 JX strain has the most homologous with A66 (DQ886445) and C80 (DQ864514) strains. By comparing with 30 strains of the same subtype published in GenBank, the homology of nucleotide sequence of different DHV-1 strains is above 94%.In the third part, according to the genome sequence of DHV-1 JX strain, we designed two pairs of primers to amplify the structure protein VP0 and VP1 genes. A BamHI site was inserted before the coding sequence of VP0 in the forward primer, and the reverse primer included a XhoI site; A EcoRI site was inserted before the coding sequence of VP1 in the forward primer, and the reverse primer included a XhoI site. VP0 and VP1 genes were amplified from the extracted RNA by RT-PCR and cloned into pET-28a(+) vector correctly. The recombinant expression plasmid pET-28a-VP0 and pET-28a-VP1 were expressed in E.coli expression system. The fusion proteins VP0 and VP1 were expressed in BL21(DE3) at a high level and confirmed by SDS-PAGE. The molecular weight of VP0 and VP1 recombinant fusion proteins were about 32 kDa and 30 kDa respectively. VP0 and VP1 fusion proteins were confirmed by Western-blot analysis with DHV-1 positive serum. VP0 and VP1 fusion proteins were mainly expressed as inclusion body.Briefly, the genome sequence of DHV-1 JX strain was determined. Comparative sequence analysis and evolutionary analysis showed that the genome sequences of DHV-1 strains from different areas have more homologous. VP0 and VP1 recombinant proteins which were expressed in prokaryotic expression system were recognized by DHV-1 antibodies in a Western-blot assay. This finding indicates that VP0 and VP1 recombinant fusion proteins can be used as antigen in enzyme linked immunosorbent assay (ELISA) of anti-DHV-1 antibody detection.