DPV UL31 Gene Prokaryotic Expression And The Study Of Its Localization And Distribution In Virus-infected Host Cells

This scientific dissertation concentrates on studies on the identified UL31 gene (Genbank accession no. EF643559) from duck plague virus (DPV). The cloning and bioinformatics analysis of the UL31 gene were studied. Furthermore, some biological characteristics, localization and distribution in virus-infected host cells of the UL31 were studied using prepared UL31 antibodies. The results are summarized as follows:1. Molecular cloning and characterization of UL31 gene from Duck enteritis virus The DPV CHv UL31 gene was composed of 933 nucleotides encoding for a polypeptide of 310 amino acid residues with an isoelectric point of 7.56; No signal peptide or any transmembrane helix was present and the subcellular localization was nucleolus. The sequence contains 28 possible sites for phosphorylation, 22 on serine, 2 on threonine, and 4 on tyrosine residues. Sequence comparison of UL31 among DPV CHv and 24 other reference herpesvirus strains showed that DPV CHv was more homologous to alphaherpesviruses than to betaherpesviruses or gammaherpesviruses. The DPV UL31sequence displayed homologies of 58.781%, 59.140% and 55.063%to the UL31 gene from Marek's disease virus type 1 (MDV-1), Meleagrid herpesvirus 1 (MeHV-1) and Bovine herpesvirus 1 (BoHV-1), respectively, suggesting a potential related function.2. DFV UL31 gene clone, expression and polyclonal antibody preparation An prokaryotic expressional plasmid pET-32-UL31 was constructed and a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 55kDa) accounted for 21.5% of total bacterial protein by gel scanning were highly expressed with 0.8mM IPTG at 37℃and found in large amounts in crude cell extracts. Purified protein was used to immunize rabbit for the UL31 anti-serum preparation. Agar diffusion reaction showed that the antibody titer was up to 1:32. Western blot analysis using the resultant sera showed that the recombinant protein was recognized by the polyclonal antibody. Thus, the polyclonal antibody prepared here may serve a good tool for study of the functional involvement of UL31 in the DPV life cycle.3. Transcriptional analysis of UL31 gene prosucts in DPV infected host cells Real-time quantitative RT-PCR was performed to determine the transcription kinetics of the DEV UL31 gene during the viral infection. The result shows the DPV UL31 gene transcripts appeared as early as 1h post infection, and then the content of transcripts increased steadily and reached a peak at 36 h, thereafter declined slowly. The result indicated that DPV UL31 gene may be a late viral gene.4. Immunolocalization of DPV UL31 gene products in virus infected DEF Indirect immunofluorescence experiments were carried out to determine the subcellular location of the UL31 protein in DEV-infected cells at different stages. The results showed that the UL31 protein was localized in the nuclear with a continuous changed form during a lytic DEV replication cycle. The specific fluorescence became first detectable at 4 h p.i, which was dispersed throughout the nuclear of infected cells. At 24 h p.i., the large amount of fluorescence was concentrated on the nucleus. After, the UL31 protein localized in very fine punctate forms dispersed throughout the nucleus of infected cells. At the late stage of DEV infection, fluorescence diminished in the nuclear, which was accumulated at the nuclear rim of infected cells. The different patterns of distribution maybe relate its important functions during egress of viral nucleocapsids from the nucleus.5. Distribution and immunolocalization of DPV UL31 gene products in virus infected duck tissues The distribution and immunolocalization of DPV UL31 gene products in virus infected duck tissues were observed and analysed using indirect immunofluorescence. The investigation showed that the DPV UL31 antigen was detected in the heart,liver,spleen,pancreas,lung,kidney,bursa of fabricius,cerebrum,thymus,duodenum,jejunum,ileum,rectum,cecal tonsil,harderian gland, but pancreas,muscles and skin shown negative. The DPV UL31 antigen can be detected from thymus,spleen and bursa of fabricius as early as 2h; the DPV UL31 antigen can be detected in most organs in 4d post infection. The results will help us to study the diagnosis and pathogenic mechanism of DPV.