Abstract
BackgroundThe duck plague virus (DPV) UL46 protein (VP11/12) is a 739-amino acid tegument protein encoded by the UL46 gene. We analyzed the amino acid sequence of UL46 using bioinformatics tools and defined the main antigenic domains to be between nucleotides 700-2,220 in the UL46 sequence. This region was designated UL46M. The DPV UL46 and UL46M genes were both expressed in Escherichia coli Rosetta (DE3) induced by isopropy1-β-D-thiogalactopyranoside (IPTG) following polymerase chain reaction (PCR) amplification and subcloning into the prokaryotic expression vector pET32a(+). The recombinant proteins were purified using a Ni-NTA spin column and used to generate the polyclonal antibody against UL46 and UL46M in New Zealand white rabbits. The titer was then tested using enzyme-linked immunosorbent assay (ELISA) and agar diffusion reaction, and the specificity was tested by western blot analysis. Subsequently, we established Dot-ELISA using the polyclonal antibody and applied it to DPV detection.ResultsIn our study, the DPV UL46M fusion protein, with a relative molecular mass of 79 kDa, was expressed in E. coli Rosetta (DE3). Expression of the full UL46 gene failed, which was consistent with the results from the bioinformatic analysis. The expressed product was directly purified using Ni-NTA spin column to prepare the polyclonal antibody against UL46M. The titer of the anti-UL46M antisera was over 1:819,200 as determined by ELISA and 1:8 by agar diffusion reaction. Dot-ELISA was used to detect DPV using a 1:60 dilution of anti-UL46M IgG and a 1:5,000 dilution of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG.ConclusionsThe anti-UL46M polyclonal antibody reported here specifically identifies DPV, and therefore, it is a promising diagnostic tool for DPV detection in animals. UL46M and the anti-UL46M antibody can be used for further clinical examination and research of DPV.
Highlights
The duck plague virus (DPV) UL46 protein (VP11/12) is a 739-amino acid tegument protein encoded by the UL46 gene
Analysis of hydrophilic and antigenic indices of the DPV UL46 protein Generally, the expression of the main antigenic regions of the protein was prioritized in order of increasing immunogenicity and specificity of the corresponding antibody
The polymerase chain reaction (PCR) products were digested with BamHI and XhoI restriction enzymes and the open reading frames (ORFs) were inserted into the pMD18-T vector to construct the cloning vectors pMD18-T/UL46 and pMD18-T/UL46M
Summary
The duck plague virus (DPV) UL46 protein (VP11/12) is a 739-amino acid tegument protein encoded by the UL46 gene. We analyzed the amino acid sequence of UL46 using bioinformatics tools and defined the main antigenic domains to be between nucleotides 700-2,220 in the UL46 sequence. The recombinant proteins were purified using a Ni-NTA spin column and used to generate the polyclonal antibody against UL46 and UL46M in New Zealand white rabbits. DPV gene transcription can be classified into 3 types: immediate-early (IE), early (E), and late (L) [12]. UL46, which is not essential for virus replication, is a late transcription gene of the herpesviruses. Generation of an antibody against DPV UL46 will further research on the function and bionomics of DPV
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