Studies On The Two-dimenshional Electrophoresis Characteristics Of The Duck Plague Virus Proteinomics And The Discovery, Prokaryotic Expression And Applications Of Duck Plague Virus UL24 Gene

Duck plague(DP),alternatively known as duck virus enteritis(DVE),is an acute, contagious and lethal septicemic disease of waterfowls such as ducks and geese in the group of Anseriformes,which can cause high morbidities and mortalities in birds and poses an important threat to waterfowl product industry.Certain DPV protein was obtained by means of two-dimensional electrophoresis and was verified by immunoblotting with the anti-DPV hyperimmune antiserum produced with the highly purified DPV preparation obtained by ultracentrifugation.The polypeptide sequences of this protein were analyzed by means of high performance liquid chromatography(HPLC) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry.Duck plague virus UL24(DPV-UL24) gene was isolated from the DPV genome by degenerative PCR with primers designed according the polypeptide sequence of the protein and verified by hybridization test.DPV-UL24 gene prokaryotic expression recombinant vector was constructed and expressed after induction.Based on the above research,the following studies were undertaken:lmmunogenicity test of the expressed DPV-UL24 protein;UL24 gene dynamic distribution and localization in DPV infected ducks by the expressed DPV-UL24 protein mediated immunohistochemistry and immunofluorescence test;Antigen capture enzyme-linked immunoabsorbent assay(ELISA) for DPV specific antibody and DPV-UL24 protein antibody capture ELISA for DPV specific antigen;Establishment of PCR detection method by DPV UL24 gene.These studies are summarized as follows:1.Ultrastructure studies showed that DEV-CHv virions have the typical morphological characteristics of herpesvirus.The cross sections of the virions were hexagonal and the mature virus with the structure of envelope,nucleocapsid and nucleus were spherical in shape with diameter of 150nm-266nm.The outer part of the envelope was somewhat densely stained than the inner part and some virions with envelop not fully formed were also observed.Most of the DEV virions have one nuclocapsids,some DEV virions have two or three nucleocapsids.The nucleocapsid of DEV was round in shape with diameter of 100nm-150nm,some densely electron-stained vires-related structures which were circle-shaped,semicircle-shaped,or U-shaped could be observed.Between the nucleocapsid and the envelop was certain electron slightly stained part;The DNA in the nucleus which was 40nm-65nm in diameter was slightly electron stained and was in cluster in appearance.Hyperimmune antiserum against DPV was produced by hyperimmunization of the mature ducks with the highly purified DPV particles.2.Research on duck plague virus proteinomics demonstrated that the following conditions favored the experiment:protein sample extracted by acetone,pH 5-8 gel strip, 30mM DTT reducing agent,carder ampholytes mixturation of loading sampling buffer,0.8 mg protein sample;1253 or 388 protein spots could be obtained respectively by coomassie brilliant blue staining and silver staining.The mean matching rate of the protein spots could reach 88.%each different time.The established model of two-dimenshional electrophoresis was stable,reliable and has a good reproducibility.At least 32 polypeptide spots could be detected by immunoblotting test and five of 32 polypeptide spots were nonspecific.The molecular weights of the 27 DPV polypeptides range from 20KD to 120 KD and the isoelectfic points range from pHS.30 to pH7.50.Two viral protein spots were selected for polypeptide sequence analysis by means of high performance liquid chromatography(HPLC) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry and seven reliable segments sequence were selected after aligning with the virus protein database.3.The discovery,prokaryotic expression,purification of products and antibody preparation of DPV UL24 gene:Two PCR products of 500 bp and 730 bp were obtained after amplifying the DPV genome with degenerative primers designed according to the polypeptide sequence.The 730 bp PCR product was verified to be duck genome specific by hybridization with DPV genome fragments processed with different restriction enzyme and forming the hybridization bands of different length.Biostatistics analysis showed that the 730 bp fragment has a maximum homology with herpesvirus genome sequence and proved that the amplified product was the DPV UL24 gene.Further analysis showed that the UL24 gene has a 1230 bp open reading flame(ORF) and is highly homologous with 11 strains of herpesvirus and is closest with Mark's virus(MDV).The UL24 protein is a conserved transmembrane protein relatively hydrophobic,with theα-helix and random coil secondary structure contents both being 46.94%.The UL24 gene with its maximum amino acids content being alanine and arginine(8.3%) encodes 409 amino acids,has 16 antigenic determinants,19 phosphorylation sites and does not have signal peptide sequence.Five potential N-glycosylation sites were spotted at the sites of 56,284,294,325 and 383 respectively.The UL24 protein is highly immunogenic,with its encoding gene amino acids codes frequency Nc value being 56.9.Twelve amino acids including alanine are of highly codon usage bias.DPV-UL24 gene was isolated from DPV genome by PCR with the specific primers designed according to the DPV-UL24 gene sequence.DPV-UL24 gene was subcloned into the vector of pMD18-T and after verification by means of PCR,restriction endonuclease restriction analysis and gene sequencing it was then cloned into the site between the restriction sites of EcoRⅠand XhoI of the pET-32a(+) prokaryotic expression vector and thus the recombinant expression plasmid of pET32a(+)/DPV-UL24 was constructed.The recombinant expression plasmid of pET32a(+)/DPV-UL24 was transformed into the host E.coli of BL21(DE3) and the approximately 38-kD UL24 recombinant protein was obtained after induction with isopropylβ- D -1-thiogalactopyranoside(IPTG),whose molecular weight basically conformed to what was predicted.Through optimization test it was found that the optimal condition was 0.2mmol/L 1PTG with an induction duration of 5 hours.The expressed protein was purified and concentrated by Ni-column affinity chromatography and after renaturation was emulsified with an equal volume of Freund's adjuvant.This product was used as vaccine to prepare hyperimmune sera with rabbits by immunization for four times.Purification of IgG fractions from antiserum was carried out by ion exchange chromatography on Econo-Pac high Q Cartridge after extraction through caprylic acid and equilibrium ammonium sulfate.4.The study on immunogenicity of prokaryotic expression product of DPV UL24 gene: The prepared UL24 recombinant vaccine was used to vaccinate 7 day old duckling and the serum collected respectively at 3,5,7,10,14,21,28,35,42,49,56,70,84 and 98 days postvaccination underwent ELISA titer and serum neutralization titer evaluation.Half of the ducks underwent challenge test 21 days postimmunization.The result demonstrated that 28 days after immunization,the greatest ELISA titer of 1:5120 at OD_450nm could be obtained in the group of recombinant protein group and the greatest ELISA titer of 1:10240 at OD_450nm could be obtained in the group of avirulent DPV vaccine group,which are both prominently higher than that of the control group(P≤0.05).Relatively high antibody levels were maintained in the two groups with a same dynamic change of decline. The antibody neutralization test showed that in the recombinant protein group and in the avirulent DPV group,neutralization antibody titer over 1:128 and 1:256 could be obtained 21 to 28 days postvaccination.The neutralization antibody and the ELISA antibody underwent a similar dynamic change in the two group.Analysis indicated that between the recombinant protein group(with its morbidity rate and mortality rate being 50%and 30% respectively) and the avirulent DPV vaccine group(with its morbidity rate and mortality rate being 30%and 10%respectively) no differences of morbidity rate and mortality rate were obtained but between the recombinant protein group and the control group(with its morbidity rate and mortality rate both being 100%) distinct differences of morbidity rate and mortality rate were obtained.All these studies demonstrated that the DPV-UL24 recombinant protein could confer duckling certain protect effects after immunization.5.The phase and location of UL24 gene express in artificial infected ducklings:The dynamic expression,infection process and distribution of DPV-UL24 gene in infected ducklings were studied by the established DPV-UL24 recombinant protein indirect immunohistochemistry method with the specimens of spleen,pancreas,harderian glands, bursa of Fabricius,liver,intestine,kidney,lung,heart,encephalon of the ducklings challenged at 28 days old with duck plague virulent DPV-CHv strain.The results demonstrated that DPV-UL24 encoding protein antigens could be detected in spleen, thymus,liver and duodenum 2 hour after challenge,in harderian glands and glandular stomach 4 hours after challenge,in ileum and lung 8 hours after challenge,in bursa of Fabricius and kidney 12 hours after challenge,in pancreas each part of intestine 24 hours after challenge.All these results demonstrated that organs of the immune system and the intestinal mucosa are the target organ and target tissue of DPV-UL24 encoding protein. DPV-UL24 encoding protein brain tissue tropism is very low,indicating that this protein is probably transmembrane protein and its function mainly related with the transportation and leading of virus components into nucleus for virion assembly during the virus proliferation.The dynamic expression,proliferation and distribution of DPV-UL24 encoding protein in infected ducklings were studied by the established DPV-UL24 recombinant protein immunofluroscence detection method in ducks challenged with duck plague virus. The results demonstrated that DPV-UL24 encoding protein antigen could be detected in spleen,thymus,harderian glands,bursa of Fabricius,liver,lung,kidney and most parts of intestine 2 hour after challenge,in pancreas,encephalon and heart 4 hours after challenge, in esophagus 8 hours after challenge.All these results demonstrated that the immune organs of spleen,thymus,harderian glands,bursa of Fabricius and the intestinal mucosa are the target organ and target tissue of DPV-UL24 encoding protein.DPV-UL24 encoding protein was selective in the target organs in the process of virus invasion and distribution, causing serious damage to mucosal epithelium.Of the 26 specimens for inspection by this method,25 specimens were positive and positive rate was 96.1%,indicating that this method for DPV-UL24 encoding protein detection is sensitive and specific.6.Research on Duck Plague Virus was Detected by Antigen Capture-ELISA with Anti-Prokaryotic Expression Protein of DPV UL24:Double antibody sandwich DPV antigen captured ELISA assay method is established and optimized with the rabbit anti-DPV UL24 prokaryotic expression protein antiserum and the duck anti- DPV UL24 prokaryotic expression protein antiserum.The results demonstrated that the optimized evaluation could be obtained when the rabbit anti DPV-UL24 antibody concentration is 5.0ug/100ul,the duck anti DPV-UL24 antibody concentration is 9.0ug/1O0ul and the enzyme linked antibody dilution is 1:2000.Other duck infected pathogens like duck hepatitis virus B(DHBV) was employed as negative controls and showed negative results. The coefficient s of variation of intra-assay and inter-assay was less than 10%and the minimum DPV content detection is 46 ng DPV purified virus,showing the specific, sensitive and stable characteristics of the established ELISA method.The dynamic proliferation and distribution of DPV-UL24 encoding protein in the specimens of liver, spleen,encephalon,esophagus,lung,and kidney of the infected ducklings were studied by this established ELISA method and the results were similar with that obtained by the established immunohistochemistry and immunoflurescence method.7.Research on Duck Plague Virus Antibody was Detected by ELISA Using Coating Prokaryotic Expression Protein of DPV UL24:Double antibody sandwich DPV antigen captured ELISA assay method is established and optimized with DPV UL24 prokaryotic expression protein as antigen.The results demonstrated that the optimized evaluation could be obtained when the DPV-UL24 recombinant protein is 80-fold diluted with its concentration being 2.5ug/100ul,the dilution of the examined serum is 1:320 and the enzyme linked antibody dilution is 1:2000.Other duck infected pathogen specific positive antiserum like duck hepatitis virus B(DHBV) and duck Riemirella Anatipestifer(RA) were employed as negative controls and showed negative results.The coefficient s of variation of intra-assay and inter-assay was less than 7%and could detect DPV positive antiserum with a dilution of 1:2560.127 specimens from the clinically infected ducklings were studied by this established ELISA method with a positive rate of 77.2%and similar results were obtained with that obtained by the whole DPV antigen as coated antigen ELISA method.8.Development and Application of PCR based on UL24 gene for the Detection of Duck plague virus:One pair of primer(P1/P2) was designed according to the DPV-UL24 genes sequence for amplification of DPV genome DNA.The results demonstrated that one PCR product of 585bp was obtained with this primers for DPV genome amplification but no products could be detected with this primer for genome amplification of other pathogen such as duck hepatitis virus and duck hepatitis virus B.The sensitivity of the primer of P1/P2 is lpg and the uniform PCR product fragment could be obtained for amplification of the 14 the specimens collected from DPV virulent virus infected ducks whereas no PCR product fragment could be obtained for amplification of the 5 the specimens collected from ducks not infected with DPV.These results demonstrated that the established UL24 PCR detection method is reliable,sensitivity and could be used in 1 the clinical diagnosis and surveillance of duck plague infection.Comparisons and analysis of the established indirect immunohistochemistry, immunofluorescent,AC-ELISA and PCR detection methods indicates that positive results could be obtained by the four methods for examination of the DPV positive specimens, specimens not infected with DPV,whereas negative results could be obtained by the four methods for examination of blank control,goat serum and other duck pathogens.These examinations indicated that these four methods are well specific.The examinations of the specimens from the artificially infected ducks indicated that positive results could be obtained in the specimens of spleen,thymus,liver and duodenum collected 4 hours postinfection.Immunohistochemistry examination indicated that UL24 gene encoding protein could be detected in almost all the tissues 24 hours postinfection,whereas UL24 gene encoding protein could be detected by immunofluorescent and PCR detection methods 12 hours postinfection.UL24 gene encoding protein could be detected in the 11 specimens by AC-ELISA methods 24 hours postinfection.The examination of the clinically infected DPV infected specimens indicated that 88.4%positive results could be obtained by immunohistochemistry method,96.1%positive results could be obtained by immunofluorescent method,85.2%positive results could be obtained by AC-ELISA methods,100%positive results could be obtained by PCR methods.