Studies On Discovery Of Duck Plague Virus GD Gene And Applications Of The Recombinant GD Protein

Duck plague (DP), also called duck virus enteritis (DVE), is an acute, contagious and lethal septicemia herpes virus infection of ducks,geese,and swans. It had causes great economical loss in domestic ducks and wild waterfowls. Duck Plague Virus (DPV), also called Duck Enteritis Virus (DEV), or Anatid herpesvirus1(AnHV-1), is an unassigned species in the FAMILY HERPESVIRIDAE. Only few research in the fields of duck plague virus genomics and gene express were performed. Based on the DPV CHv DNA gene library constructed in our laboratory, DPV glycoprotein D (gD) gene sequence was first identified (Gene Bank Accession No. EU195085), a series of studies were conducted and results got as follows:l.The discovery, Cloning and Molecular Characterization of duck plague virus gD gene According to the CHv strain of duck plague virus (DPV CHv) DNA gene library constructed in our laboratory, A complete open reading frame (ORF1269) found was homology to herpes virus glycoprotein D gene. Sequence analysis confirmed that the ORF1269is DPV gD gene. Bioinformatics analysis showed that DPV gD gene is1269bp in length, encoded a422amino acid. Its molecular weight was45kD and the isoelectric point (PI) was6.66. DPV gD gene is closer to Felid herpesvirus1(FeHV-1), Phocid herpesvirus1(PhoHV-1) and Canid herpesvirus1(CaHV-1) than others in genetic relationship, while gD is closer to Gallid herpesvirus1(GaHV-1). Signal peptide located at positions laa-22aa. While transmembrane helix at7aa-29aa and360aa-382aa. There are4N-linked glyeosylation sites,9O-linked glyeosylation sites and25Phosphorylation site in DPV gD. The main location site in cell is endoplasmic reticulum, about44.4%, and the others are cytoplasm, vacuolar, extracellular, including cell wall, mitochondrial and nuclear, about11.1%each.2. Cloning, prokaryotic expression, purification and antibody preparation of Duck plague virus gD gene According to the Duck plague virus gD gene sequence found, Extracellular region of DPV gD gene about729bp was amplified and cloned into pGEM-T vector. After confirmed with restriction enzyme digestion and DNA sequencing. The gD gene fragment were sub cloned into Msc I and HindⅢ site of prokaryotic expression vector pET-32a (+).Recombinant plasmid pET-32a-gD was constructed successfully. Plasmid PET32a-gD was transformed into competent host bacteria E. coli BL21(DE3). After induction with IPTG, size of approximately30kD recombinant protein was detected by SDS-PAGE electrophoresis。Recombinant protein expressed as two forms: the soluble and insoluble inclusion body. The results of Western blotting demonstrated that the protein can specifically react with anti-DPV serum. The recombinant gD protein was purified by using Ni-NTA affinity chromatography. Immunized rabbit with purified gD extracellular region protein mixed with Freund’s adjuvant three times, the ELISA method was used to measure the titers of anti-gD antibody in the serum. The titer is1:256000. Collected the immuned rabbit serum, anti-gD IgG was purified by using Protein A affinity chromatography,3. Construction of Duck plague virus gD gene eukaryotic expression vector and its transient expression in COS-7cells According gene sequence of DPVgD, a pair of specific primers was designed, the whole gene sequence was amplified from the DPV genome. Insert the fragment between the cloning sites NheⅠ and HindⅢ of the eukaryotic expression vector pEGFP-N1and pcDNA3.1(+), then the Recombinant eukaryotic expression plasmid pEGFP-N1-gD and pcDNA3.1(+)-gD were successfully constructed. then the Recombinant eukaryotic expression plasmids were transfected in COS-7cells Liposome-mediated method, using laser scanning confocal microscope,Western-blotting, indirect immunofluorescence method were used to observe the expression of gD protein. The fluorescent protein punctate aggregated in cytoplasm and cell membrane of COS-7transfected plasmid pEGFP-Nl-gD, mainly located in the cell membrane. Data of Western-blotting showed molecular weight of the gD expressed in COS-7cells was about55KD, a little larger than molecular weight of gD predicted. The fluorescent protein punctate aggregated in cytoplasm,nuclease and cell membrane of COS-7transfected plasmid pcDNA3.1(+)-gD,mainly located in the cell membrane.4.The transcription and expression analysis of duck plague virus gD and the subcellular localization of gD protein in the DPV-infected DEF cellsDEF cells prepared as conventional methods used. CHv strains inoculated whenthe cells grow as a monolayer. Samples were collected respectively after inoculated Oh (blank control group),2h,4h,6h,8h,12h,24h,48h,60h. and dealed with(1) extracting total cellular RNA,(2) extracting total cellular proteins,(3) fixing cell. Then used the sample for (1) detecting gD gene transcription phase by Fluorescent quantitative PCR,(2) detecting gD gene expression phase by Western-blotting,(3) detecting gD subcellular localization of gD protein by indirect immunofluorescence method. The results show that the gene has begun to transcript from2h after DPV infection,8h began to express. Transcription and expression increased rapidly after12h then continued to60h. The expression product of gD gene in the DEF cells appeared high abundance, molecular weight of mainly mature glycoprotein is55kD. the subcellular localization of gD protein in DEF cells is a dynamic process. localized in the cytoplasm from8h after DPV infection, after12h in nuclear membrane, after24h in cell membrane, after48h in nuclear membrane, the cell membrane and cytoplasm. Continued to60h, the location is irregular because of cell fusion and disintegration. The location feature just the same herpes virus glycoprotein, Confirmed the similarity of DPVgD function with other herpes virus gD function.5. Establishment and Application of an Indirect Recombinant Glycoprotein D-based Enzyme-linked Immunosorbent Assays (ELISA) An indirect ELISA for detecting antibody of duck plague was developed using purified Recombinant gD as antigen. anti-DPV sera were used to carry ELISA.Data of chessboard titration showed that the fitful concentration of antigen is100ng per well (1μg/mL,100μL/well), and of serum, is1:80. Results of crossing test and interdiction test demonstrated specificity and sensitivity of the method. The right coating condition is in37℃1h, then4℃overnight. 5g/L gelatine PBST is the fitful block solution. The indirect gD-ELISA was specific and sensitive according to the detection of DHV, DHBV and GpV. Statistical data of90clinical samples showed that there was no difference between two detection systems: gD-ELISA and DPV-ELISA. The recombinant protein gD has high immunoreactivity. gD-based indirect ELISA method can be used in diagnosis of DPV in clinic.6Development and application of a Polymerase Chain Reaction to Detect DPV gD gene According to the DPV gD gene fragment lack of signal peptide, a PCR assay was developed. A specific DNA fragment about1009bp was obtained from DNA of DPV CHv strain and DPV vaccine. There are no specific fragments were amplified from normal DEF,duck embryo allantoic fluid, duck hepatitis virus, duck hepatitis B virus and gosling plague virus., at the same time,some brands varies in length can be amplified from DNA of herpes virus HSV-1,HSV-2and BV, demonstrated that gD is Congeneric in herpes virus homology. The length of the amplified fragment is not the same as the gD gene expected, indicating the specificity of the PCR primers and methods. Susceptibility test results show that about lpg DPV DNA can be detected. Application of the PCR method of to detect, the right length and specific fragment can be amplified from DPV infected tissue liver, spleen, thymus, lymph nodes and Bursa of Fabricius.The positive bands were not detected in the normal tissues. Confirmed that the method can be used in clinical detection of DPV.