DPV UL26.5 Gene Prokaryotic Expression And The Study Of Its Cellular Localization In Virus-infected Cells

A pair of specific primer was designed and synthesized according to the duck plague virus UL26.5 gene (GenBank NO. EF643564) identified in author's lab. Then PCR amplification, sequencing and bioinformatics analysis of DPV UL26.5 gene were studied. Furthermore, prokaryotic expression and polyclonal antibody were prepared, and transcriptional analysis and time course of gene products in DPV infected host cells were established. The results were as follows:1. Molecular characteristics of DPV UL26.5 geneThe DPV UL26.5 gene was composed 1074bp encoding for a polypeptide of 357 amimo acid residues. The amino acid sequences predicted from the nucleotide contained six conserved functional domains, a hydrolysis site of serine protease, some phosphorylation site which related to the function of UL26.5 protein. The protein was no transmembrane helices and hydrophilic. The phylogenetic tree demonstrated that DPV UL26.5 gene was related to UL26.5 of poultry herpes virus (alphaherpesvirinae) more closely.2. DPV UL26.5 gene prokaryotic expression and polyclonal antibody preparationThe amplified fragment was inserted into pMD18-T vector, then the pMD18/UL26.5 and pET-32a were digested with BamH I and Xho I. The UL26.5 gene was sub-cloned into prokaryotic vector pET-32a. A single positive colony was picked and identified by PCR and restriction analysis. Then the recombinant plasmid pET32a-UL26.5 was transformed into E. coli BL21(DE3) strain and expressed under IPTG induction. SDS-PAGE analysis showed that the induced expressed protein was about 59ku. The proteins were highly expressed with 0.2 mmol/L IPTG at 37℃for 4 h, and found in large amounts in crude cell supernatant. 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. Transcriptional analysis of UL26.5 gene in DPV infected host cells The transcription analysis of UL26.5 gene in DPV infected cells was detected by FQ-PCR. The results showed the UL26.5 gene transcripts slowly rised up first, then sharply rised up with the extension of the infection time, at last lowed down slowly. The UL26.5 gene products appeared low level before 12hpi, then the signal intensity increased steadily and reached a peak at 54 hpi, and remained detectable up to 72 hpi, which owes the characterization of herpervirus late genes. The transcription of DPV UL26.5 gene suggests that the gene characteristics and function, as well as the proliferation of virus, could be the important factor.4. Expression and cellular localization of DPV UL26.5 protein in virus infected DEFImmunolocalization detection was observed using immunofluorescence technique, results shown that specific fluorescence appeared in cell nucleus as early as 10 hours post infection. With the extension of the infection time, the more specific fluorescence appeared in the nucleus, and the point fluorescence gradually gathered to the nuclear membrane. This change in distribution can be UL26.5 protein of DPV to form scaffold in the nucleus, to further promote nucleocapsid assembly and release from the internal core of the capsid.