Studies On Some Characteristics Of Duck Plague Virus UL51 Gene And Applications Of The Recombinant UL51 Protein

1 Sequence analysis and codon bias analysis of duck plague virus UL51 gene According to duck plague virus (DPV) DNA library constructed in our laboratory, and combining together with the analysis of open reading frame (ORF) Finder tool and BLAST tool of NCBI, a complete ORF (GenBank accession number DQ072725) of DPV was identified as a tegument protein encoding the DPV UL51 gene with a size of 759 bp, and contains a conserved domain of Alphaherpesvirinae UL51. Then, phylogenetic tree analysis revealed that the DPV UL51 gene was evolutionarily closer to Mardivirus genus of the Alphaherpesvirinae subfamily. After that, the UL51 gene was analyzed by a series of bioinformatics tools. The results showed that the DPV UL51 protein contained fourteen potential phosphorylation sites, one potential palmitoylation site and four potential linear B-cell epitopes, suggesting that it might be a phosphorylated and palmitoylated protein, and be able to induce a very strong immune response. Meanwhile, the DPV UL51 protein does not contain any N-linked glycosylation sites, transmembrane helix, signal peptides, and nuclear location signals (NLS), but it contains Golgi apparatus location signals, suggesting that it might localize to the Golgi apparatus of the cytoplasm. Besides, the results of codons usage bias analysis of the DPV UL51 gene showed that, codon of the gene was strong bias towards the synonymous codons with A and T at the third codon position, and the prokaryotic expression system might be more suitable for the expression of the gene. These results provided theoretical supports for the further study on the characterization and function of DPV UL51.2 Cloning, prokaryotic expression, purification and polyclonal antibody preparation of DPV UL51 gene A pair of primers was designed based on the DPV UL51 gene sequence identified by our laboratory. The UL51 gene fragment was amplified from the genome of DPV by PCR, and cloned into pMD18-T vector and sequenced. Then the gene from the pMD18-UL51 vector with two restriction enzymes (EcoR I and XhoⅠ) digestion was subcloned into the prokaryotic expression vector pET-28a (+) to generate the recombinant plasmid pET28-UL51, which was identified by two restriction enzymes (EcoR I and XhoⅠ) digestion, and then transformed into E. coli BL21(DE3) strain and expressed under isopropylβ-D-1-thiogalactopyranoside (IPTG) induction. SDS-PAGE analysis showed that the induced expressed protein is about 34 KD, and mostly exists in inclusion examined by soluble analysis. Through optimization test it was found that the optimal condition was 0.8mmol/L IPTG as inductor, duration of 4 hours at 37℃. The fusion protein was purified by Ni2+-NAT column affinity chromatography, and used to immunize rabbits to generate the UL51 antiserum. Its antibody titer tested by agar gel precipitation was up to 1:32. The UL51 antibody IgG was subsequently obtained by using caprylic acid and ammonium sulfate precipitation and High-Q anion-exchange chromatography. These researches will provide a basis for further functional analysis of the DPV UL51 gene.3 The transcription and expression characteristics of DPV UL51 gene in DPV-infected cells In this study, we determined the transcription and expression characteristics of DPV UL51 gene by real-time quantitative RT-PCR and western blotting. The results indicated that DPV UL51 gene transcripts appeared at 2 h post infection (PI), and its expression products were first detected at 8 h PI, and then both of the transcripts and expression products were up to a peak at 48 h PI, thereafter both of them were reduced; A 34 KD protein in DEF cells was detected, and the UL51 protein was a component of DPV virions. This study provides serviceable datum for elucidating characteristics and functions of the DPV UL51 gene.4 Subcellular localization of DPV UL51 protein in DPV-infected host cells The results of indirect immunofluorescence technique showed that specific fluorescence appeared in cytoplasm as early as 8 hour PI and a great deal of specific fluorescence concentrated in the juxtanuclear region from 24 to 36 h PI,but there after the specific fluorescence became to weaken followed by the appearance of cytopathic effect. Transmission immunoelectron microscopy analysis revealed that DPV protein (pUL51) was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism. In a word, these experimental results lay a foundation for further study on the function of DPV UL51 protein.5 Construction of the DPV UL51 gene eukaryotic expression vector and transient transcription and expression of the gene in COS-7 cells Based on the sequence of DPV UL51 gene to design a pair of primers, the gene was amplified by PCR, and cloned into pMD18-T vector. After restriction enzyme digestion and sequence analysis, it was subcloned into the pcDNA3.1 eukaryotic expression vector to generate the recombinant plasmid pcDNA3.1-UL51, which was transfected into the COS-7 cells by Lipofectin method; then, its transcription, expression and localization were determined by real-time qualiticative RT-PCR, western blotting and indirect immunofluorescence. The results indicated that its transcripts appeared at 6 h post transfection (PT), and its expression products were first detected at 12 h PT, and then both of the transcripts and expression products were up to a peak at 24 h PT, thereafter both of them were reduced; A 33 KD protein in COS-7 cells was detected, and the UL51 protein was localized to the perinuclear regions at 12 h PT, and to the nucleus and cytoplasm at later times. This study provides serviceable datum for elucidating characteristics and functions of the DPV UL51 gene.6 The localization and dynamic distribution properties of DPV UL51 protein in experimentally DPV-infected ducks detected by immunohistochemistry staining Fifty-eight 30-day-old DPV-free ducks were intramuscularly inoculated with the pathogenic DPV CHv strain as infection group, and two ducks were selected as pre-infection group.18 different tissues (bursa of Fabricius, thymus, spleen, Harders glands, liver, pancreas, esophagus, glandularis ventriculus, duodenum, jejunum, ileum, caecum, rectum, cerebrum, kidney, lung, myocardium, muscle) were collected from DPV-infected ducks at sequential time points, and prepared for indirect immunofluorescence staining and immunoperoxidase staining. The results showed that in acute DP cases, DPV pUL51 was mainly distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus and intestine, and was mostly localized in the cytoplasm of lymphocytes, reticulum cells, macrophages and epithelial cells. The research will be not only useful for understanding the pathogenesis of this DP, but also for studying the localization and distribution properties of alphaherpesvirus pUL51 homologs.7 Development and application of an indirect ELISA and an immunochromatographic strip test based on recombinant UL51 protein for detecting antibody against DPV DPV infection causes substantial economic losses to the worldwide duck-producing areas. The monitoring of DPV-specific antibodies is a key to evaluate the effect of weak-DPV vaccine and develop rational immunization programs. Thus, in this study, based on a purified recombinant UL51 protein, an indirect ELISA (UL51-ELISA) and an immunochromatographic strip (UL51-ICS) test were developed for detecting the DPV serum antibodies. The optimum conditions for UL51-ELISA were determined. The results demonstrated that the optimized evaluation could be obtained when the recombinant UL51 protein concentration is 2.5μg/100μL, the dilution of the examined serum is 1:200, and the enzyme linked antibody dilution is 1:2000. Other duck infected pathogen specific positive antiserum, such as duck hepatitis virus (DHV), duck riemirella anatipestifer (RA) and duck E.coli, were employed as negative controls and showed negative results. The coefficients of intra-assay and inter-assay variation were less than 10% and could detect DPV positive antiserum with a dilution of 1:3200. The UL51-ICS test is based on membrane chromatography, and uses both the recombinant UL51 protein conjugated with colloidal gold and goat anti-rabbit IgG conjugated with colloidal gold as the tracers. In the UL51-ICS, the purified recombinant UL51 protein was used as the capture reagent at the "test line", and the purified rabbit IgG was used as the capture reagent at the "control line". The optimum conditions for UL51-ICS test were determined. The results demonstrated that the optimized evaluation could be obtained when the recombinant UL51 protein concentration is 2mg/mL, the rabbit IgG concentration is lmg/mL, the recombinant UL51 protein conjugated with colloidal gold concentration is 2mg/mL, and the goat anti-rabbit IgG conjugated with colloidal gold concentration is 2mg/mL. Other duck infected pathogen specific positive antiserum were employed as negative controls and showed negative results. The reproducibility of intra-assay and inter-assay were good. The strips were stable for one year at 4℃or 25℃, and could detect DPV positive antiserum with a dilution of 1:128. To evaluate the effect of the UL51-ELISA and the UL51-ICS test,110 duck serum samples collected from several duck flocks were simultaneously tested by the UL51-ELISA, UL51-ICS, the whole DPV antigen as coated antigen ELISA method (DPV-ELISA) and neutralization test (NT). The results of detections revealed that, compared with the DPV-ELISA and NT, both the UL51-ELISA and UL51-ICS test have higher specificity, higher sensitivity, highest coincidence, low cost, and are suitable for the serological surveillance of DPV infection in the field or in the laboratory.8 Development and application of an antigen capture ELISA method and an immunochromatographic strip test based on anti-recombinant UL51 protein polyclonal antibody for detecting DPV antigen An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) and an immunochromatographic strip (ICS) test were developed with the purified rat anti-UL51 recombinant protein polyclonal antibody and rabbit anti-UL51 recombinant protein polyclonal antibody for detecting DPV. For AC-ELISA, the 1:640 diluted viruses in the DPV-infected cells can be observed, while other pathogens such as DHV, RA, E.coli can not be detected by the AC-ELISA. For ICS, the 1:80 diluted viruses in the DPV-infected cells can be observed, while other pathogens such as DHV, RA, E.coli can not be detected by the AC-ELISA, too. Ten cloaca cotton pledget samples of ducks which were experimentally infected with virulent DPV CHv, were assayed. The results of detections revealed that, compared with the PCR, both the AC-ELISA and ICS test have higher specificity, higher sensitivity, higher coincidence, and can be used to diagnose DPV in the field or in the laboratory.