Cloning,Expression,Polyclonal Antibody Preparation And Prelimary Applieation Of Duck Plague Virus UL21Gene

Directed by Professor:CHENG Anchun and WANG MingshuThe study focuses researches on bioinformatics analysis of gene sequence, cloning, expression, protein purification and polyclonal antibody preparation of duck plague virus UL21gene, real-time fluorescent quantitative reverse transcription PCR analysis of gene transcription phase, the main results are as follows:1. Bioinformatic analysis of UL21gene of duck Plague VirusThe whole UL21gene of DPV is1686bp length, and the product consists of561amino acids with prediction premolecular weight62KD and pI value5.49. The UL21protein contain27potential phosphorylation sites,3potential glycosylation sites an both the signal peptide and the transmembrance region are not found, he protein subcellular localization mainly locates at cytoplasmic with65.2%, nuclear with17.4%. The phylogenetic tree and amino acid sequence comparison analysis revealed DEV UL21protein is conserved and most closely related to Varicellovirus and Mardivirus. t codon usage bias of DEV UL21had strong bias towards the A-ended or T-ended codons.2. Cloning, expression, polyclonal antibody preparation of UL21gene of duck Plague VirusWe designed a pair of primers using primer5tool to clone the whole UL21gene according to the DPV UL21gene sequence. The amplified gene fragment is1735bp length(containing whole UL21ORF), and the product was sent to Takara to constructe the PGEMT-UL21T cloning plasmid. We also constructed the recombinant plasmid pET32(c)-UL21and expressed in E. coli B21system. We determined the pUL21best expression conditions:30°C,0.4mmol/L IPTG final concentration induced expression of the8hours by optimizing the temperature, IPTG concentration and the expression of time. The protein was separated by SDS-PAGE, and recovered to purify by cutting gel. We immuned rabbits to prepare polyclonal antibody and the highest antiserum titer reached1:16by agar gel diffusion test.3. Analyzing transcription phase of duck plague virus UL21gene and the subcellular localization of UL21proteinThe transcription phase was analysed by SYBR Green I real-time fluorescence quantitative RT-PCR. We found that the UL21gene is firstly detected at36h after the cell was infected. Then the transcription increased and reached the peak at72h, and then began to decrease the level of transcription.. After inoculation of the cell culture solution was added to a working concentration of ganciclovir, the UL21gene transcription was inhibited proved the DPV UL21is a late gene.