DPV U_L26 Gene Prokaryotic Expression And The Study Of Its Cellular Localization In Virus-infected Host Cells

This article has carried out series researches on the newly discovered U_L26 gene (assigned accession no.EF643565) of the duck plague virus(DPV) by the means of sequence characterization and bioinformatics analysis,cloning,prokaryotic expression, preparation of polyclonal antibody,time course of gene products in DPV infected host cells by real-time PCR and expression phase analysis by westem blotting.Results were reported as following:1.Sequence characterization of the DPV U_L26 geneThe DPV U_L26 gene was composed of 2124 nucleotides encoding for a polypeptide of 707 amino acid residues.Through the prediction and analysis of signal peptide, transmembrane domain,phosphorylation site and hydrophobicity,found U_L26 had not signal peptide and transmembrane domain,had as much as 55 phosphorylation sites,and the protein is a hydrophilic one.Cladogram analysis found U_L26 had belong toα-Herpesvirus, which was relationship with GaHV-2,GaHV-3,MeHV-1.2.DPV UL26 gene clone,prokaryotic expression and polyclonal antibody preparationAn prokaryotic expressional plasmid pET-32/U_L26 was constructed and a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 97kDa) were highly expressed with 0.2mM IPTG at 30℃under IPTG induction until 6h,and it may found in large amounts in supernatant.Purified protein was used to immunize rabbit for the U_L26 anti-serum preparation.The titre of U_L26 anti-serum examined by agar diffusion test is 1:32.3.Immunolocalization of DPV U_L26 gene products in virus infected DEFFluorescence through the cell-mediated immunity to virus infection of the subcellular localization DEF testing,The results showed that specific fluorescence appeared in nucleus as early as 8 hours post infection and a great deal of specific fluorescence concent rated in the cell nucleus by 18 hours.But after 40 hours,the fluorescence in some part of one nucleus was dispersed.Fifty-six hours later,the fluorescence weakened significantly in all cell nucleus,and it appeared nuclear division and different morphology of nucleus.4.Time course of gene products in DPV infected host cells and transcriptional analysisTime course of transcriptional is a method of detecting gene transcription by real-time PCR using relative quantitative.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 38h,but after this point it became to decrease,we can detect the mRNA expression until 70h.The result of gene products in different time is:UL26 protein can be detected from 12h,its expression level to the maximum among 40h and deduced from 56h.The results of two time course analysis showed that the law of UL26 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 UL26 gene is a late gene from our research.