Prokaryotic Expression Of The Small Subunit Of Ribonucleotide Reductase (R2) Gene From Duck Plague Virus And Its Transcription And Expression Dynamic Analysis

Duck plague (DP) is caused by duck plague virus (DPV), which is a herpesvirus. DP is an acute, contagious, and fatal epidemics in the family Anseriformes (such as ducks, geese, and swans). In this study, bioinformatics analysis, recombination proteins expression and polyclonal antibodies preparation, and transcription and expression dynamic analysis of the small subunit of ribonucleotide reductase (R2) gene (Accession No.:EU071039) from DPV-CHv strain (which was registered to NCBI GeneBank from Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University). The results are as follows:1. Bioinformatics analysis of DPV R2gene. The DPV R2gene was analysed by bioinformatics. The results show that the encoding protein of this gene, comprising355amino acids with a theoretical molecular weight of40.68kDa, is an unstable, slightly acidic, and hydrosoluble protein, which contained19phosphorylation sits,7glycosylation site, a transmembrane domain, but without signal peptide. The subcellular localization analysis finds that this protein mainly locates at cytoplasmic and nuclear. Moreover, the results of amino acid sequence similarity and phylogenetic evolution relation analysis of DPV R2protein indicate that there is a high homology between DPV and the viruses of Mardivirus genus of the subfamily Alphaherpesvirinae.2. Recombination protein expression and polyclonal antibody preparation of DPV R2gene. On the basis of DPV R2gene sequence, a pair of primers was designed and used to amplify the R2gene. According to the results of bioinformatics analysis of DPV R2gene and the experience of expression of herpesviruses R2gene, the recombination protein DPV R2was expressed by the pET32a(+) prokaryotic expression system on low temperature (25℃), low concentration of IPTG (0.2mmol/L), and long induced time (12h). The results of SDS-PAGE and Western Blotting analysis show that the recombination protein is about64kDa, and can react with anti-DPV polyclonal antibodies. And then, the purified recombination protein was used to prepare polyclonal antibody. The agar gel diffusion test results indicate that the titer of prepared polyclonal antibody is1:32.3. Analysis of transcription and expression dynamic of DPV R2gene. On the basis of DPV R2gene sequence, a pair of primers was designed and used to analyze the transcription dynamic of R2gene. The result of drug inhibition test indicates that DPV R2gene is an early gene; and the results of real-time fluorescent quantitation PCR analysis and Western Blotting indicate that the transcription and expression dynamic of R2gene is firstly detected at1hour post infection (hpi), after that, the transcription level become sustainable growth with the time, moreover, the transcription level reached a peak at56hpi, then maintained a high level.