Discovery, Prokaryotic Expression And Applications Of Duck Plague Virus GC Gene

Duck plague(DP),also called duck virus enteritis(DVE),is an acute,contagious and lethal septicemic herpesvirus infectionn of ducks,geese,and swans.It had causesed great economical loss in domestic ducks and wild waterfowls.Only few researches in the field of duck plague virus genomics had been performed.According to DPV gC gene sequence discovered by our laboratory,a series of scientific research were conducted and the results were obtained as followed:1.Discovery,Cloning and Molecular Characterization of duck plague virus gC gene According to the duck plague virus(DPV) DNA gene library constructed in our laboratory,A complete open reading frame(ORF1296) discovered was homology to other herpesvirus glycoprotein C gene.Sequence anylysis and Dot-blot hybridization confirmed that the ORF1296 is DPV gC gene(GenBank accession number is EU076811).Bioinformatics analysis showed that DPV gC gene is 1296bp in length,encoded a 432 amino acid.Its molecular weight was 45kD and the isoelectric point(PI) was 5.292.Duck plague virus gC gene is more close genetic relationship to gallid herpesvirus than to other's gC gene.Amino acids sequcence at positions 21～24,50～58,67～70,161～171,273～281,387～393 maybe the most possible antigenic epitopes.2.Prokaryotic expression,purification and antibody preparation of Duck plague virus gC gene According to the Duck plague virus gC gene sequence found by our laboratory,DPV gC gene was amplified and cloned into pMD18-T vector.It was identified by PCR,restriction endonuclease digestion analysis and DNA sequencing.The gC gene was subcloned into EcoRI and XhoI site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-gC was constructed successfully.The plasmid was transformed into host bacterium of BL21(DE3) induced with IPTG and analyzed by SDS-PAGE electrophoresis.the expected 65kD fusion protein was expressed.Western Blot test showed that the expressed fusion protein reacted to DPV serum was positive.Then rabbits were immuned with the extracted,purified pET32a-gC expressed products.The rabbit-anti DPV gC protein polyclonal antibodies were purified and proved to have good immunogenicity.3.Imunogenicity of Duck plague virus gC gene prokaryotie products The DPV gC prokaryotic expression products were purified,renatured and prepared as vaccine with Freund'S adjuvant to ducklings.The results showed that recombinant protein can induce neutralization antibody anti DPV,its titer reached the maximum 21 days after immuned in accordance with attenuated vaccine group and its variation law of antibody titers detected by ELISA was coincided with attenuated vaccine group.There was no difference in the immune protective efficiency between the recombinant protein groups and attenuated vaccine group.All shows that DPV gC gene recombinant protein has certain protective capacity to virulent DPV challage.4.Development and aplication a SP assay and an indirect immunofluorescence assay to detect Duck Plague Virus gC protein Rabbit anti-DPV gC IgG purified by ammonium sulfate precipitation method and High Q anion exchange column chromatography were used as the first antibody,a streptavidin-peroxidase conjugated method and an indirect immunofluorescence assay was established respectively.DPV antigens distribute,dynamic distribution in spleen,thymus, brain,bursa of Fabricius,liver,lung,kidney and intestine challenged DPV CHv strain and expression kintics of DPV gC gene were researched by the two methods.The results showed that DPV was one kind of pantropic virus,spreaded through blood to all kinds of tissues.The expression kintics was that 8 hour after challenge 12h,live,esophagus,glandular stomach were dectected gC protein expression.24h after infected,bursa of Fabricius,kidney and spleen were dectected gC protein;2d 1,gC proteins were dectected in lung and brain and 3d later,expressed protein were dectected in almost infect tiusses.5.Development and application of an AC-ELISA with Anti-Prokaryotie Expression Protein of DPV gC By using recombinant gC expressed protein as the primary antibody and purified Rabbit anti-DPV IgG as the second antibody,an antigen capture ELISA(AC-ELISA) to dectect DPV was developed and optimized.The results showed that the optimal concentration of the recombinant gC protein was dilution of 1:40,Rabbit anti-DPV IgG was dilution of 1:80 and HRP-goat anti rabbit IgG dilution of 1:2000.No cross reaction was observed with DI-IBV,DHV,GPV,R.A.,E.coli,S.anatum and the minimum DPV content detection is 30 ng DPV.In the intro-batch duplicativity test and in the inter-batch duplicativity test,the variation coefficient is less than 10%.6.Establishment and Aplication of an Indirect ELISA Diagnose with the DPV gC expresssion protein An indirect ELISA for detecting antibody of duck plague was developed using recombinant gC expressed in E.coli as coating antigen.The optimum conditions for the gC-ELISA were determined.The results showed that the concentration of the recombinant gC protein was 1.6μg for coating per well;the optimal dilution of the examined serum is 1:80 and the enzyme linked antibody dilution is 1:1000.The indirect gC-ELISA was specific and sensitive and used in diagnosis of DPV in clinic.7.A PCR method based on DPV gc gene was established to Detect DPV According to the DPV gC gene,a PCR asssy was developed.The specific DNA fragment(about 1112bp) was amplified from DPV DNA,but not from nucleic acid samples of DHV,DHBV,GPV,E.coli, S.anatum,R.A.The specific DNA product was also amplified from the infected livers and brain from 19 samples of DPV.These results suggested that the PCR was a specific and sensitive method which could be used for detection and diagnosis of clinical DPV infection.8.Comparison of the four assays to detect Duck Plague Virus In order to select more rapid,sensitive and specific method in detection of DPV directly from the clinical specimens, tissues infected DPV were detected with AC-ELISA,SP immunohistochemistry,indirect immunofluroscence and PCR.The results showed that the sensitivity of the PCR much higher than the AC-ELISA,and the AC-ELISA is higher than the SP and IF method.The remits to detect clinical samples showed that there were no significant difference in the positive rates among the four methods.