The Prokaryotic Expression, Protein Purification, Time Courses Of Transcription And Expression And Subcellular Localization Of DPV UL35 Gene

This article has carried out series researches on the identified UL35 gene(assigned accession no.EF643558) 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 the DPV UL35 gene DPV UL35 gene was composed of 354 nucleotides encoding for a polypeptide of 117 amino acid residues. Its encoding protein(VP26) had a Herpes_UL35 conserved domain related with small nucleocapsid protein family and highly conserved among the Herpes UL35 proteins. Moreover,the nucleic acid and amino acid sequence of DPV VP26 had higher homology with UL35 homologous protein of Alphaherpesvirus than others.Phylogenetic tree analysis showed that on taxonomic status the DPV could be grouped in Alphaherpesvirus subfamily and,might be a branch of Alphaherpesvirus,that was a branch of Alphaherpesvirus subfamily of waterfowl.Besides,subcellular location analysis demonstrated that the VP26 mainly located in nucleus and was a nuclear-targeted protein. Condon preference analysis demonstrated that the alternative codons for the same amino acid in VP26 had distinctly different frequency and the VP26 had 18,19 and 25 codons' frequency evidently disagreeing with E.coli,yeast and human,respectively.Thus,it could be concluded that codon preference of DPV VP26 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 UL35 gene' expression.2.Cloning,prokaryotic expression,preparation of polycional antibody of DPV UL35 gene The amplified fragment of DPV UL35 gene was inserted into pMD18-T vector,then the pMD18-T-UL35 and pET-32a(+) were digested with BarnHⅠand HindⅢ, the UL35 gene was subcloned into prokaryotic vector pET-32a(+).After identification of the recombinant plasmid pET-32a(+)-UL35 by PCR and restriction digestion of BamHⅠand HindⅢ,the recombinant plasmid was used to transform into E.coli BL21(DE3) competent cell.By IPTG-inducible expression,a polypeptide with a size corresponding approximately to that expected for the recombinant protein(about 33kDa) accounted for 32.3%of total bacterial protein by gel scanning were highly expressed with 1.0 mmol/L IPTG at 34℃for 5 h,and found in large amounts in crude cell extracts.The expression product was purified by passing the Ni+ affinity chromatograph collumn using the recombinant protein with a tag of 6×His.The rabbit was immunized with the purified protein,and agar diffusion reaction showed that the antibody titer was up to 1:32.3.Time courses of transcriptional and expressional analysis of UL35 gene products in DPV infected duck embryo fibroblasts The transcriptional analysis result of UL35 gene products in infected duck embryo fibroblasts(DEF) detected by FQ-PCR showed the transcriptional products of DPV UL35 gene sharply rised up first,then lowed down slowly with the extension of the infection time.The transcription of DPV UL35 gene could be detected at the early time of 0.5 h post infection(hpi),followed by rapid amplification of the transcriptional products to the peak.Sequently,the transcriptional products lowed down to a certain degree at 24 hpi but maintained at a relative high level at 72 hpi,which owes the typical characterization of herpervirus late gene.A time course of expression of DPV infected DEF was analyzed by western blot using the purified polyclonal antibody IgG against DPV VP26.A specific immunoreactive band migrating was observed at the expected position for protein DPV VP26(about 13kDa),with maximal amount at 72 hpi and remained gradual increase among 12 to 48 hpi.There,it can be presume from the aggregate analysis of time courses model of transcription and expression that the DPV UL35 gene is a late gene,the magnanimous expression of DPV UL35 in the late infection stage suggests that DPV VP26 may closely related to the assembly and maturation of DPV.However,its precise role may depend on its further research.4.Subcellular localization analysis of DPV UL35 gene Subcellular localization detection of virus-infected DEF using immunofluorescence technique showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 h and increased gradually in 12 to 36 h and eventually reached to the maxlmum,which aggregated in the spot region of the nucleus after the DEF were infected by DPV.However,there were only a small amount of specific fluorescences in the cytoplasm in 12 h and increased with the extension of infection time in 24 to 48 h.Then,the specific fluorescences finally reached to the maxlmum in the cytoplasm in 72 h.So,it can be infered from the distribution characteristics that DPV VP26 was synthetized in cytoplasm then transported to nucleus for the assembly of capsid precursor for the execution of its biological function.