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

This article has carried out series researches on the newly discovered dUTPase gene (assigned accession no. DQ486149) of the duck plague virus (DPV) by the means of sequence characterization and codon usage bias analysis, cloning, prokaryotic expression, preparation of polyclonal antibody, time course of gene products in DPV infected host cells and transcriptional analysis. Also, indirect immunofluorescence (IFA) and indirect immunohistochemistry (IIS) were established on the base of dUTPase protein for the detection of DPV in paraffin-embedded tissues and the dynamic monitoring of dUTPase gene products localization distribution in DPV infected duck tissues. Results were reported as following:1. Sequence characterization and codon usage bias analysis of the DPV dUTPase gene The DPV dUTPase gene was composed of 1344 nucleotides encoding for a polypeptide of 447 amino acid residues. Moreover, the dUTPase of DPV has five conserved motifs(motifl PKRTEDAAYDI,motif2 GRSS,Motif3 GLIDSGYRG,motif4 GDRIAQ,motif5 RQFSGFGS) as that among dUTPase family proteins, and the arrangement according to 3-1-2-4-5 belongs to the ClassⅡdUTPase subfamily. Sequence comparison of dUTPase among DPV and 33 other reference herpesvirus strains showed that DPV CHv was more homologous to alphaherpesviruses than to betaherpesviruses or gammaherpesviruses. Sixty-one codons in the predicted polypeptide, with a strong bias towards A and T at the third codon position, were used. Comparison of the codon usage in the dUTPase gene of different organisms reveals that there are 19 codons showing distinct codon usage differences between the DPV and Escherichia coli dUTPase genes; 16, between the DPV and yeast dUTPase genes; and 15, between the DPV and human dUTPase genes Analysis of variance (ANOVA) showed significant differences between the DPV and yeast dUTPase genes (r = 0.536, P < 0.01).2. DPV dUTPase gene clone, prokaryotic expression and polyclonal antibody preparation By constructting the prokaryotic expressional plasmid pET-32-DUT, a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 66.8kDa) accounted for 36.2% of total bacterial protein by gel scanning were highly expressed with 0.8mM IPTG at 30℃, and found in large amounts in crude cell extracts. Purified protein was used to immunize rabbit for the dUTPase anti-serum preparation. Its ELISA antibody titer was up to 1:409600. High specific IgG polyclonal antibody was obtained by purification using the caprylic acid and ammonium sulfate precipitation and High-Q anion-exchange chromatography.3. Time course of gene products in DPV infected host cells and transcriptional analysis RNA Dot-blot shows the DPV dUTPase gene transcripts appeared as early as 30 min post infection, then the hybridization signal intensity increased steadily and reached a peak at 4 hpi, and remained detectable up to 72 hpi, which owes the typical characterization of herpervirus early genes. A time course of DPV infected DEF were analyzed by western-blot using the polyclonal antibody IgG against DPV dUTPase. A specific immunoreactive band migrating was observed at the expected position for protein DPV dUTPase (about 49kDa), with maximal amounts between 12 and 24 hpi and remained detectable in late of infection. Aggregate analysis the time course model of transcriptional and translational, it can be presume the DPV dUTPase gene is a early gene, the expression of DPV dUTPase may play an activation role in the late expression of capsid protein and peplos protein. And, it is close correlated to the virus vegetative cycle.4. Immunolocalization of DPV dUTPase gene products in virus infected DEF Immunolocalization detection was observed using immunofluorescence technique, results shown that specific fluorescence appeared in cytoplasm as early as 4 hours post infection and a great deal of specific fluorescence concentrated in the cell nucleus by 12 hours. But after 24 hours, the fluorescence in nucleus was dispersed while the cytoplasm increased. Forty-eight hours later, the fluorescence weakened significantly in both cytoplasm and nucleus. This distribution feature can primary infer that the DPV dUTPase synthesized in cytoplasm, then changed into the nucleus to enduce the base biological function, catalyzes the conversion of dUTP to dUMP and PPi, decreases Uracil incorporated in DNA synthesis and reduces cells dUTP / dTTP ratio to ensure the accuracy and smoothly of DNA replication.5. Distribution and immunolocalization of DPV dUTPase gene products in virus infected duck tissues The Distribution and immunolocalization of DPV dUTPase gene products in virus infected duck tissues were observed and analysed using indirect immunofluorescence and indirect immunohistochernistry techniques. The investigation showed that the DPV dUTPase antigen was detected in the heart,livert,spleen,pancreas,lung,kidney,bursa of fabricius,cerebrum,thymus,duodenum,jejunuim,ileum,rectum,cecal tonsil,harderian gland, but trachea,muscles,skin shown negative. The DPV dUTPase antigen can be detected from cerebrum,cecal tonsil and bursa of fabricius as early as 2hpi. And the DPV dUTPase antigen positive detection rate in 12hpi was more than 50 percent and later reached a peak at 4 days post infection. It is mostly located in the extremity differentiated cells such as nerve cells, hepatocyte, immunity organ lymph-macrophage. Thus, once the ducks infected with DPV, nervous system and immune organ were first attacked, then to damage the tissues all over the body. The virus encodes a great quantity DPV dUTPase in earlier period of virus infection. It is possible to compensate the cells dUTPase insufficient which is induced by the insufficient and cells functional incapacitation, to ensure the accuracy and smoothly of DNA replication and provide powerful guarantee to make virus tissue pantropic infection all over the body.