DPV UL32 Gene Prokaryotic Expression And The Study Of Its Subcellular Localization In Virus-infected Host Cells

This article has carried out series researches on the identified UL32 gene (assigned accession no. EF643561) of the duck plague virus (DPV) in our laboratory by the means of molecular characteristics analysis, cloning, prokaryotic expression, preparation of polyclonal antibody, time course of gene products in DPV infected host cells and transcriptional analysis. Results were reported as follows:1. Molecular characteristics analysis of DPV UL32 gene DPV UL32 gene was composed of 1962 nucleotides encoding for a polypeptide of 653 amino acid residues. The production of DPV UL32 gene had a Herpes_env conserved domain related with Herpesvirus putative major envelope glycoprotein family. The phylogenetic tree demonstrated that DPV UL32 gene was related to UL32 of poultry herpes virus (alphaherpesvirinae) more closely. Through the prediction and analysis of signal peptide, transmembrane domain, phosphorylation site and hydrophobicity, found DPV UL32 had not signal peptide and transmembrane domain, had as much as 33 phosphorylation sites, and the protein is a hydrophilic one. Condon preference analysis demonstrated that the alternative codons for the same amino acid in DPV UL32 gene had distinctly different frequency and the DPV UL32 had 24, 28 and 29 codons' frequency evidently disagreeing with E.coli, yeast and human, respectively. Thus, it could be concluded that codon preference of DPV UL32 gene was close to that of the prokaryote and deviated from eukaryote and human. Prokaryotic expression system such as E.coli system should be more suitable for DPV UL32 gene' expression.2. DPV UL32 gene clone, prokaryotic expression and polyclonal antibody preparation By constructting the prokaryotic expressional plasmid pET-32a-UL32, a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 92.2kDa) accounted for 33.1% of total bacterial protein by gel scanning were highly expressed with 1.0mM IPTG at 25℃, and found in large amounts in crude cell extracts. Purified protein was used to immunize rabbit for the DPV UL32 protein anti-serum preparation. and agar diffusion reaction showed that the antibody titer was up to 1:16. High specific IgG polyclonal antibody was obtained by purification using the caprylic acid and ammonium sulfate precipitation and High-Q anion-exchange chromatography.3. Expression and subcellular localization of DPV UL32 protein in virus infected DEF Immunolocalization detection was observed using immunofluorescence technique, results shown that specific fluorescence appeared in cytoplasm as early as 10 hours post infection and the specific fluorescence can be dected in the nucleus after 16 hours post infection. After 24 hours post infection, the fluorescence accumulating in regions which was recognized as the packaging sitere, in the nucleus adjoin the trachychromatic region, the role of UL32 protein is involved in efficient localization of capsids to replication compartments . The fluorescence was appeared homogeneous distribution in the nucleus after 48 hours after infection.4. Time courses of transcriptional and expressional analysis of UL32 gene products in DPV infected host cells We can find the increase of target gene's mRNA expression as early as 10h by SYBR Green I dye method detecting cell samples from different time,and reached its maximum at 60h, but after this point it became to decrease, at 70 hours after infection, we can still detecting a high level of transcriptional, with the value of 278. The result of gene products in different time is: UL32 protein can be detected from 12h, its expression level to the maximum at 60h and can be detced deduced at 72h. The results of two time course analysis showed that the law of UL32 gene' s transcription and expression generally conformed to the gene's life circle regulation from transcription to translation, moreover it satisfied the model that transcription is the former one and expression is later after it. We can summarize that DPV UL32 gene is a late gene from our research.