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Prokaryotic Expression of UL2Protein of Duck Plaque Virus Strain and a PCR Detection Method to Identify DPV Virulent Strain and Attenuated Vaccine Strain

Author: YinXueQin
Tutor: ChengAnChun
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Duck plague virus UL2gene prokaryotic expression antibody preparation antidiastole PCR
CLC: S858.32
Type: Master's thesis
Year: 2013
Downloads: 17
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Abstract


Little studies on DPV UL2gend have been reported.This essay focused on DPV UL2gene and UL2protein’s bioinformatics analysis, prokaryotic expression, polyclonal antibody preparation. And based on the UL2gene sequence characteristics, a PCR method to identify the affection of DPV virulent strain and attenuated vaccine was designed.1. Bioinformation analysis of sequence characteristics and codon bias of duck plague virus UL2protein. UL2gene from duck plague virus (DPV) encodes uracil-DNA glycosylase (UDG). Bioinformatics analysis was performed to predict the characteristics of uracil-DNA glycosylase, the results indicated that UL2gene encodes a polypeptide, molecular mass of37.2946Kda and comprising333amino acids, without signal peptide and the transmembrane region. And though multiple sequence alignment, four conserved structural motifs, eighteen functional sites and many antigenic determinants were found. And UL2gene had no more contiguous rare codon.2. Cloning, prokaryotic expression, purification and polyclonal antibody preparation of DPV UL2gene. Based on the UL2gene sequence, a pair of primer were designed by Primer Premier5.0. DPV UL2gene was amplified and the product was cloned into the multiple cloning site of the pMD18-T vector. A prokaryotic expression vector pET32a(+) was used to product recombinant protein. pMD18-UL2was digested with BamH1and Hind III and UL2sequense was inserted into the BamH Ⅰ/Hind III site of pET-32a(+) and the recombinant plasmid pET32a-UL2was transformed into Rosseta for recombinant protein production, SDS-PAGE showed that the induced expressed protein is about58kDa. Using Ni-NTA method to purify recombinant protein and the purified protein was used to immunize rabbits four times for the preparation of polyclonal antibody. The highest antibodies titer was1:32.3. A detection method to identify DPV virulent strain and attenuated vaccine strain by polymerase chain reaction targeting the UL2gene The sequence analyses find that the UL2gene between DPV virulent strain and attenuated vaccine strain is different. The UL2gene length of virulent strain is1002bp, but the UL2gene fragment of commercial vaccine strains is474bp. Based on the difference, a PCR method was designed to identify the infection. The specificity and sensitivity assay show this method is effective and useful for clinical diagnosis between DPV virulent strain and attenuated vaccine strain.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Duck
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