Studies On The Discovery, Prokaryotic Expression And Applications Of Duck Plague Virus US3 Gene

1 Discovery,Clone and Molecular characterization of Duck Enteritis Virus US3 gene Duck enteritis virus(DEV) causes substantial duck farms losses,however,its molecular biology is poorly understood.Here,an open reading frame of an US3-1ike gene of DEV was identified from a DEV genomic library.Its existence was confirmed by cloning from DEV-infected duck embryo fibroblasts(DEFs) and DNA sequencing.In addition,an US3 protein phylogenetic tree was constructed and showed that the evolutionary relationship of DEV is close to the genus Mardivirus.Bioinformatic analyses of the DEV US3-like protein predicted its post-translational modifications,secondary structure and tertiary structure,and suggested that the protein contains a serine-threonine kinase domain.2 Prokaryotic expression,purification and polyclonal antibody preparation of US3 gene of duck enteristis virus The recombinant plasmid pMD18T-US3 was successfully constructed as the methods in the last chapter.The recombinant plasmid pMD18T-US3 by two restriction endonucleases BamH I and Xho I was subcloned into the polyclonal sites of the plasmid pET-32a(+) digested by the same restriction endonucleases to construct recombinant expression plasmid vector pET32a-US3.After the plasmid pET32a-US3 was conformed into the host E.coli of BL21(DE3),the bacteria was induced by IPTG(β-D-1-thiogalactopyranoside) to express a recombinant protein with approximately 63kD of molecular weight.Further by optimizing the expression system,an optimal condition was verified as the recombinant expression induced by 0.6 mmol/L IPTG for 4 h at 30℃.The expressed protein was subject to purifying through Ni-column affinity chromatography.The purified recombinant protein was emulsified with an equal volume of Freund's adjuvant to vaccine rabbit for preparing anti-US3 sera.The anti-US3 IgG was purified from the sera by ion exchange chromatography combined with caprylic acid and equilibrium ammonium sulfate method.3 The transcript phase,translation phase and subcellular localization of the duck enteritis virus US3 gene The transcript phase of DEV US3 gene was detected by real-time quantitative PCR.The results showed that the gene began to transcript at 1 h post infection and was up to the peak at 8 h post infection while the levels of transcript were obvious to fall after 12 h post infection.In contrast,the transcript of US3 gene fluctuated at a lower level at 24 h,48 h and 60 h post infection than at 8 h post infection.In addition,the expression phase of DEV US3 gene was detected by use of indirect immunofluorescent assay.The results showed that no expression of the gene at 1 h post infection was detected and up to 2 h post infection,a weak immunofluorescence was able to detect.The expression of the US3 gene was more and more accompanied by the increasing infection period and the expression product of US3 gene was full of the cytoplasm and perinuclear area at 12 h post infection and 24 h post infection.In the subcellular localization assay,the results showed that the US3 protein was mainly localizated around the nulear area at 2 h post infection,4 h post infection and 8 h post infection.At 12 h and 24 h post infection,the protein was mainly localized at cytoplasm.4 Development and Application of an Indirect Immunohistochemistry Staining Method Based on the Antibody against Recombinant Prokaryotic Expression protein of Duck Enteritis Virus US3 Gene A indirect immunohistochemistry staining method based on the antibodies against DEV US3 protein was successfully constructed to dectect the DEV US3 protein.The method was verified to be specific to the DEV US3 protein through detecting the tissues infected by different viruses which include DEV,DHV, Riemerella anatipestifer and DHBV.In the further inspection on the clinical specimens infected by DEV by this method,all the 20 specmens were identified to be positive.The rate of detection is 100%.In addition,the ducklings were challenged by DEV strain CHv and the different tissue specimens,which included heart,liver,spleen,lung,kidney, thymus gland,bursa,brain,glandular stomach,pancreas,dodecadactylon,jejunum, harderian glands,esophagus and rectum,were collected respectively at 2h,4h,6h,8h, 12h,24h,2d,3d,5d,7d after infection.Expressions of DEV US3 protein at the different tissues were dectected by use of the indirect immunohistochemistry staining method to study the dynamic distribution of US3 protein in the tissues.The result showed that the DEV US3 protein was dectected in heart,liver,spleen,lung,kidney,thymus gland, bursa at 6 h post infection,in brain,glandular stomach,pancreas,harderian glands, dodecadactylon and jejunum at 8 h post infection and in esophagus and rectum at 12 h post infection.5 Development and Application of an Indirect Immuno-Fluorescent Staining Method based on the Antibody against Recombinant Prokaryotic Expression protein of Duck Enteritis Virus US3 Gene A indirect immuno-fluorescent staining method based on the antibodies against DEV US3 protein was successfully constructed to dectect the DEV US3 protein.The method was verified to be specific to detect the DEV US3 protein through detecting the tissues infected by different viruses which include DEV,DHV, Riemerella anatipestifer and DHBV.In the further detection on the clinical specimens infected by DEV by this method,all the 20 specmens were identified to be positive.The rate of diagnosis is 100%.In addition,the ducklings were challenged by DEV strain CHv and the different tissue specimens,which included heart,liver,spleen,lung,kidney, thymus gland,bursa,brain,glandular stomach,pancreas,dodecadactylon,jejunum, harderian glands,esophagus and rectum,were collected respectively at 2h,4h,6h,8h, 12h,24h,2d,3d,5d,7d after infection.Expressions of DEV US3 protein at the different tissues were dectected by use of the indirect immunohistochemistry staining method to study the dynamic distribution of US3 protein in the tissues.The result showed that the DEV US3 protein was dectected in thymus gland,bursa at 4h post infection,in heart,liver,spleen,lung,brain,pancreas,kidney at 6h post infection,in glandular stomach, harderian glands,esophagus,rectum dodecadactylon and jejunum at 8h post infection.6 Research on Detection of the Antibody against Duck Enteritis Virus by indirect ELISA Using Prokaryotic Expression Protein of DEV US3 as Coating Antigen Based on the Prokaryotic Expression Protein of DEV US3 gene as coating antigen,an indirect ELISA was developed for detection on the antibodies against DEV.In the research,the optimum conditions for ELISA were determined.The results showed that the optimal concentration of the coating antigen was 2.2μg/mL and the optimal dilution of the examined serum is 1:320.In addition,under the optimal condition of concentration of the coating antigen and dilution of the examined serum,the optimal dilution of the enzyme linked antibody was determined as 1:2000.The method was verified to be specific to DEV by detecting the serums aganst different viruses such as DEV,duck hepatitis virus B (DHBV) and duck riemirella anatipestifer(RA) etc.The sensitivity of the method was up to 1:6400.In the repeat experiments,the variation coefficients in intra-assay and inter-assay were less than 5%.In further,120 clinical specimens from the infected ducklings were detected by this indirect ELISA method and the positive rate is 85%which is consistent with the result by use of the indirect ELISA method based on DEV as coating antigen.7 Development and Application of PCR based on DEV US3 gene for the Detection of Duck plague virus Based on the DEV US3 gene which was first found in this paper,a pair of primers was designed to dectect the DEV by use of PCR.The results showed that a anticipated product with 148 bp was amplified.In further,the PCR method was verified to be specific to amply DEV DNA but not to amplify the DNAs from duck hepatitis virus,duck hepatitis virus B,GPV,R.A,E.coli and S.anatum.The sensitivity of the PCR method is 3 fg.In addition,by use of the PCR method 15 different tissues from ducks infected by DEV and 3 different tissues from ducks uninfected by DEV were detected and the results showed that all the tissues from the ducks infected by DEV were amplified to produce a 148 bp product but there were nothing to amplify in all the tissues from the ducks uninfected by DEV.In conclusion,this PCR method was sensitive and specific to DEV DNA and was able to be used in clinical detection.