Primary Studies On The Function Of Duck Plague Virus Ge Gene

1. Bioinformation analysis of sequence characteristics and codon bias of duck plague virus gE gene. DPV gE gene was 1473bp, and the GenBank accession number was EU071044. The characteristics of the protein encoding DPV gE gene were analyzed by the bioinformatics software, the results showed that DPV gE gene was encoded a protein comprising 490 amino acids, which contained an N-terminal signal peptide,21 antigenic determinants,4 potential palmitoylation sites,29 phosphorylation sites, and 6 glycosylation sites. DPV gE was a type-I membrane protein that could be resolved into 3 distinct functional domains:a 396-amino-acid extracellular domain, a 23-amino-acid hydrophobic transmembrane domain, and a 71-amino-acid cytoplasmic domain. Subcellular location analysis demonstrated that gE mainly located in the cytoplasm, and the phylogenetic tree analysis showed that DPV gE was evolutionarily closer to the mardivirus genus of the Alphaherpesvirinae subfamily. Meanwhile, the codons usage bias analysis revealed that DPV gE was strong bias towards the synonymous codons with A and T at the third codon position. And we should choose the host bacteria, which should impove the expression of the exogenous genes, if gE was expressed by using the prokaryotic expression system.2. Cloning, prokaryotic expression and polyclonal antibody preparation of DPV gE gene. The primers were designed based on the sequence of DPV gE by Primer Premier 5.0. DPV gE gene was amplified from the genome of DPV by PCR, and cloned into pMD18-T vector, which was identified by restriction enzymes digestion (EcoR I and Xho I) and sequenced. The correct recombinant vector was named pMD18-T-gE. Then gE gene from the pMD18-T-gE vector with two restriction enzymes digestion was subcloned into the prokaryotic expression vector pET-32a (+) to generate the recombinant plasmid pET32-gE. And the recombinant plasmid pET32-gE was transformed into E. coli BL21(DE3), BL21(pLysS) and Rosseta strain, expressed by IPTG (isopropylβ-D-thiogalactopyranoside) induction, and the analysis of the expression conditions was optimized, which contained the expressed host strans,the different induction time and temperature. The result showed that the optimal condition was 0.2mmol/L IPTG as inductor, duration of 4.5 hours at 30℃, and SDS-PAGE analysis showed that the induced expressed protein is about 74 KD, and the recombinant protein was mostly existed in inclusion examined by soluble analysis, and purified by washing inclusion body, and used to immunize rabbits for the preparation of polyclonal antibody, which was subsequently obtained by using ammonium sulfate precipitation and High-Q anion-exchange chromatography, and the pET32a/DPV-gE antiserum had a high level of specificity.3. Cellular localization of DPV gE protein in DPV-infected cells. The cellular localization of DPV gE protein was tested with the anti-DPV polyclonal IgG as the first antibody by indirect immunofluorescence analysis. The result showed that specific fluorescence was first appeared in cytoplasm at 5.5h PI (post infection), and the fluorescence was stronger from 9 to 24h PI gradually. At 36h PI, these fluorescence granules was detected widely distributed in the cytoplasm, and became more bigger and brighter. The gE-specific fluorescence was gradually diminished at 48h PI with the cytopathic effect (CPE), and a deal of the specific fluorescence was concentrated in the juxtanuclear region. Then at 60h PI, the gE-specific fluorescence was sparser and weaker following the cytoplasm disintegration in infected cells, when the DEFs cells were exfoliated and the plaques size become bigger.4. The transcription and expression characteristics of DPV gE gene in DPV-infected host cells. The transcription and expression characteristics of DPV gE gene were determined by real-time quantitative PCR and western blotting. These results revealed that the transcripts of DPV gE were appeared at 4 h post infection (PI), and its expression products were detected at 8h PI, and the transcripts and expression products were up to a peak at 36h PI, thereafter both of them were reduced. And the molecular weight of expression products was approximate 54 KD in lysates of DPV-infected cells.5. The distribution of DPV gE protein in experimentally DPV-infected ducks detected by indirect immunoperoxidase assay (IPA).30-day-old ducks were intramuscularly inoculated with DPV CHv strain, and the different tissues were collected from DPV-infected ducks at sequential time points. And the tissues were tested with the polyclonal pET32a/DPV-gE IgG as the first antibody by indirect immunoperoxidase assay (IPA). The result showed that DPV gE was first detected in the immunological organs (Bursa of Fabricius, thymus and spleen) at 6h PI (post infection), then it appeared in the Harders glands, macrophages, glandularis ventriculus, liver and intestine at 8h-12h PI, and the intensity of positive staining in various tissues increased steadily, finally, in the kidney, lung, myocardium and cerebrum at 24h-48h PI.6. The distribution of DPV gE protein in experimentally DPV-infected ducks detected by indirect immunofluorescence assay (IFA).30-day-old ducks were intramuscularly inoculated with DPV CHv strain, and the different tissues were collected from DPV-infected ducks at sequential time points. And the distribution of DPV gE on DPV-infected ducks was detected with the polyclonal pET32a/DPV-gE IgG as the first antibody by indirect immunofluorescence assay (IFA). DPV gE was distributed in the immunological organs (spleen, Bursa of Fabricius, thymus, Harders glands), digestive organs (liver, intestine, esophagus, glandularis ventriculus) and other parenchymatous organ (kidney, myocardium, cerebrum, lung). And DPV gE was first seen in the BF and spleen at 4h PI (post infection), then it was detected in Harderian gland, thymus, liver and the intestine at 8h PI, and it was shown in the kidney, lung, myocardium, and cerebrum 12h PI. And the positive fluorescent signals increased from 12h to 216h PI.7. Development and application of an indirect ELISA based on the recombinant protein pET32a/DPV-gE for detecting the antibody against duck plague virus. Based on the purified recombinant protein pET32a/DPV-gE, DPV-gE-ELISA was developed for detecting the DPV serum antibodies. And the optimum conditions for DPV-gE-ELISA were determined, and the results showed that the optimized concentration of the recombinant protein pET32a/DPV-gE is l:100(2μ.g/100μl), the enzyme linked antibody dilution is 1:1000, and the dilution of the examined serum is 1:160. Duck S.anatum, duck E.coli, duck riemirella anatipestifer (RA) and duck hepatitis virus (DHV) were employed as negative controls, and detected by DPV-gE-ELISA, and the result was negative. The coefficients of intra-assay and inter-assay variation were less than 10%, and DPV-gE-ELISA could detect DPV positive antiserum with a dilution of 1:1280. To evaluate the effect of the DPV-gE-ELISA,55 duck serum samples collected from several duck flocks were simultaneously tested by the DPV-gE-ELISA and the whole DPV antigen as coated antigen ELISA method (DPV-ELISA). The results revealed that DPV-gE-ELISA have higher coincidence compared with the DPV-ELISA (about 87.27%).