Key Laboratory of Animal Diseases and Human Health of Sichuan Province

Yaan, China

Key Laboratory of Animal Diseases and Human Health of Sichuan Province

Yaan, China
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Hu X.,Sichuan Agricultural University | Hu X.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | Wang M.,Sichuan Agricultural University | Wang M.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | And 16 more authors.
Poultry Science | Year: 2017

The UL13 protein of the duck enteritis virus (DEV), predicted to encode a Ser/Thr protein kinase, belongs to the family of conserved herpesvirus protein kinases (CHPK), which plays an important role in herpesvirus proliferation. In this study, truncated UL13 was expressed as a fusion protein of approximately 44 kDa using a prokaryotic expression system, and this protein was used to generate a specific anti- UL13 antibody. This antibody detected UL13 starting at 4 h post infection in duck embryonic fibroblast cells and identified UL13 to be present in both the cytoplasm and the nucleus. UL13 RNA was found to be transcribed starting at 2 h post infection, and the synthesis of the UL13 mRNA was found to be sensitive to the protein synthesis inhibitor cycloheximide (CHX) and tolerant of the DNA polymerase inhibitor ganciclovir (GCV). Its nuclear location and status as an early gene suggested that DEV UL13 might play important roles in DEV replication, which was confirmed by comparing the proliferation of a UL13-knockout mutant virus, a revertant virus, and the parent virus in cell culture. The specific mechanisms of UL13 in viral replication need to be further studied. © 2017 Poultry Science Association Inc.


Cao X.,Sichuan Agricultural University | Cheng A.,Sichuan Agricultural University | Cheng A.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | Wang M.,Sichuan Agricultural University | Wang M.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province
Proceedings - 4th International Conference on Computational and Information Sciences, ICCIS 2012 | Year: 2012

This report showed some physico-chemical properties and structural features about UL41 protein predicted by some software and on-line tools. The analysis showed both the signal peptide and the trans membrance region are not found. Meanwhile, the protein has twenty-four potential phosphorylation sites. In addition, the protein has more hydrophilic amine acid districts than hydrophobic districts and subcellular localization mainly located at the cytoplasm with 56.5%. The prediction of secondary structure showed that random coils dominate among secondary structure elements followed by alpha helix and extended strand. The prediction of tertiary structure showed the structure of the UL41 protein had a certain level of similarity to the structure-specific nuclease FEN-1(flap endonuclease-1). The study will be a basis for the further functional study of the UL41 protein. © 2012 IEEE.


Cao X.,Sichuan Agricultural University | Cheng A.,Sichuan Agricultural University | Cheng A.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | Wang M.,Sichuan Agricultural University | Wang M.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province
Proceedings - 4th International Conference on Computational and Information Sciences, ICCIS 2012 | Year: 2012

The Duck Plague Virus (DPV) is the causative agent of Duck plague, which causes significant economic losses in the commercial duck industry all over the world. Our laboratory had identified and sequenced the DPV UL41 gene. Bioinformatics analysis of DPV UL41 gene were performed by some software and online tools. The results showed that GC content of duck plague virus UL41 gene was 42.42%, which encoded a protein with 498 amino acids in the peptide. The UL41 gene sequence didn't contain a rare codons string with more than two rare codons. The construction of phylogenetic tree suggested DPV had a close evolutionary relationship with the branch of the mardivirus genus, including MeHV-1, GaHV-2 and GaHV-3. © 2012 IEEE.


Chang H.,Sichuan Agricultural University | Cheng A.,Sichuan Agricultural University | Cheng A.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | Wang M.,Sichuan Agricultural University | And 10 more authors.
Virology Journal | Year: 2010

Background. The gE protein of duck plague virus is the important membrane glycoprotein, its protein characterization has not been reported. In this study, we expressed and presented the characterization of the DPV gE product. Results. According to the sequence of the gE gene, a pair of primers were designed, and the DNA product with 1490bp in size was amplified by using the polymerase chain reaction (PCR). The PCR product was cloned into pMD18-T vector, and subcloned into pET32a(+), generating the recombinant plasmid pET32a/DPV-gE. SDS-PAGE analysis showed that the fusion pET32a/DPV-gE protein was highly expressed after induction by 0.2 mM IPTG at 30°C for 4.5 h in Rosseta host cells. Over expressed 6×His-gE fusion protein was purified by nickel affinity chromatography, and used to immunize the rabbits for the preparation of polyclonal antibody. The result of the intracellular localization revealed that the gE protein was appeared to be in the cytoplasm region. The real time PCR, RT-PCR analysis and Western blotting revealed that the gE gene was produced most abundantly during the late phase of replication in DPV-infected cells. Conclusions. In this work, the DPV gE protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gE product for the first time. These properties of the gE protein provided a prerequisite for further functional analysis of this gene. © 2010 Chang et al; licensee BioMed Central Ltd.


Xiang J.,Sichuan Agricultural University | Ma G.,China Rural Technology Development Center | Zhang S.,Sichuan Agricultural University | Cheng A.,Sichuan Agricultural University | And 10 more authors.
Virology Journal | Year: 2010

Knowledge of the intracellular location of a protein can provide useful insights into its function. Bioinformatic studies have predicted that the DEV pUL38 mainly targets the cytoplasm and nucleus. In this study, we obtained anti-pUL38 polyclonal sera. These antibodies were functional in western blotting and immunofluorescence in DEV-infected duck embryo fibroblasts (DEFs). pUL38 was expressed as a 51-kDa protein from 8 h post-infection onward, initially showing a diffuse distribution throughout the cytoplasm, and later in the nucleus. Furthermore, pUL38 was found in purified virus. These results provide the first evidence of the kinetics of expression and intracellular localization of DEV pUL38. © 2010 Xiang et al; licensee BioMed Central Ltd.


Chanjuan S.,Sichuan Agricultural University | Anchun C.,Sichuan Agricultural University | Anchun C.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | Mingshu W.,Sichuan Agricultural University | And 14 more authors.
Avian Diseases | Year: 2010

To determine the expression and distribution of tegument proteins encoded by duck enteritis virus (DEV) UL51 gene in tissues of experimentally infected ducks, for the first time, an immunoperoxidase staining method to detect UL51 protein (UL51p) in paraffin-embedded tissues is reported. A rabbit anti-UL51 polyclonal serum, raised against a recombinant 6-His-UL51 fusion protein expressed in Escherichia coli, was prepared, purified, and used as primary antibodies. Fifty-eight 30-day-old DEV-free ducks were intramuscularly inoculated with the pathogenic DEV CHv strain as infection group, and two ducks were selected as preinfection group. The tissues were collected at sequential time points between 2 and 480 hr postinoculation (PI) and prepared for immunoperoxidase staining. DEV UL51p was first found in the spleen and liver at 8 hr PI; in the bursa of Fabricius and thymus at 12 hr PI; in the Harders glands, esophagus, small intestine (including the duodenum, jejunum, and ileum), and large intestine (including the caecum and rectum) at 24 hr PI; in the glandularis ventriculus at 48 hr PI; and in the pancreas, cerebrum, kidney, lung, and myocardium at 72 hr PI. Thúroughout the infection process, the UL51p was not seen in the muscle. Furthermore, the intensity of positive staining of DEV UL51p antigen in various tissues increased sharply from 8 to 96 hr PI, peaked during 120144 hr PI, and then decreased steadily from 216 to 480 hr PI, suggesting that the expressional levels of DEV UL51p in systemic organs have a close correlation with the progression of duck virus enteritis (DVE) disease. A number of DEV UL51p was distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine, and large intestine of DEV-infected ducks, whereas less DEV UL51p was distributed in the Harders glands, glandularis ventriculus, cerebrum, kidney, lung, pancreas, and myocardium of DEV-infected ducks. Moreover, DEV UL51p can be expressed in the cytoplasm of various types of cells, especially most abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of UL51p expression and distribution in vivo but also for a greater understanding of the pathogenesis of this DVE. © 2010 American Association of Avian Pathologists.


Xiang J.,Sichuan Agricultural University | Zhang S.,Sichuan Agricultural University | Cheng A.,Sichuan Agricultural University | Cheng A.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | And 11 more authors.
Virology Journal | Year: 2011

Background: Previous studies have indicated that the VP19c protein and its homology play similar roles in capsid assembly of all Alphaherpesvirus subfamily. However, there is no report on the VP19c protein of duck enteritis virus (DEV). In this study, we expressed the DEV VP19c protein and presented its antigenic properties. Moreover, we developed polyclonal antibody against the VP19c protein and characterized it. Methods. A recombinant VP19c (rVP19c) and N-terminal were expressed in Escherichia coli (E.coli) and purified by Ni2+-affinity chromatography. The antigenic properties of the recombinant protein were determined by Western blot and indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the polyclonal antibodies against the purified recombinant proteins were produced and the titer of polyclonal antibody was determined by ELISA analysis. Finally, the antibody was used to recognize the VP19c in the cells infected with DEV in the immunofluorescence assay. Results: The N-terminally His-tagged rVP19c and rVP19c(N) were produced as inclusion bodies in E. coli strain BL21 (DE3) with molecular weight of about 66 and 46 kDa. Then the proteins were purified to reach the level of homogeneity. Western blot and ELISA analysis that the rVP19c seems to be structurally and antigenically very similar to native VP19c and the N-terminus of VP19c may contain most antigenic linear-epitopes. Furthermore, ELISA analysis demonstrated that the titer of polyclonal antibody was approximately 1:12800, and in the immunofluorescence assay, the antibody was able to recognize the VP19c in the cells infected with DEV. Conclusions: To our knowledge, this was the first report on basic properties of DEV VP19c protein. In the present study, we obtained a high-level expression of the recombinant VP19c protein as well as high titers of rabbit polyclonal antibody against to VP19c protein. The anti-rVP19c serum was able to detect the VP19c protein in DEV infected cells and the VP19c protein targeted to the nucleus as distinct punctate speckles. This specific polyclonal antibody provides a good tool for further studying structural and functional characterization of DEV VP19c. © 2011 Xiang et al; licensee BioMed Central Ltd.


Lian B.,Sichuan Agricultural University | Xu C.,Sichuan Agricultural University | Cheng A.,Sichuan Agricultural University | Cheng A.,Key Laboratory of Animal Diseases and Human Health of Sichuan Province | And 12 more authors.
Virology Journal | Year: 2010

Background. Viral envelope proteins have been proposed to play significant roles in the process of viral infection. Results. In this study, an envelope protein gene, gC (NCBI GenBank accession no. EU076811), was expressed and characterized from duck plague virus (DPV), a member of the family herpesviridae. The gene encodes a protein of 432 amino acids with a predicted molecular mass of 45 kDa. Sequence comparisons, multiple alignments and phylogenetic analysis showed that DPV gC has several features common to other identified herpesvirus gC, and was genetically close to the gallid herpervirus. Antibodies raised in rabbits against the pET32a-gC recombinant protein expressed in Escherichia coli BL21 (DE3) recognized a 45-KDa DPV-specific protein from infected duck embryo fibroblast (DEF) cells. Transcriptional and expression analysis, using real-time fluorescent quantitative PCR (FQ-PCR) and Western blot detection, revealed that the transcripts encoding DPV gC and the protein itself appeared late during infection of DEF cells. Immunofluorescence localization further demonstrated that the gC protein exhibited substantial cytoplasm fluorescence in DPV-infected DEF cells. Conclusions. In this work, the DPV gC protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gC product for the first time. These properties of the gC protein provided a prerequisite for further functional analysis of this gene. © 2010 Lian et al; licensee BioMed Central Ltd.


PubMed | Key Laboratory of Animal Diseases and Human Health of Sichuan Province and Sichuan Agricultural University
Type: | Journal: Virology journal | Year: 2015

The UL54 protein of Duck Enteritis Virus (DEV) is a homolog of herpes simplex virus-1 (HSV-1) immediate-early infectious cell protein 27 (ICP27), a multifunctional protein essential for viral infection. Nonetheless, there is little information on the UL54 protein of DEV.The UL54 gene was cloned into the pPAL7 vector, and the recombinant protein, expressed in the E. coli Rosetta, was used to produce a specific antibody. Using this antibody, Western blotting and indirect immunofluorescence analysis (IFA) were used to analyze the expression level and intracellular localization, respectively, of UL54 in DEV-infected cells at different times. Real-time quantitative reverse transcription PCR (RT-PCR) and the pharmacological inhibition test were utilized to ascertain the kinetic class of the UL54 gene.UL54 was expressed as a fusion protein of approximately 66.0 kDa using the prokaryotic expression system, and this protein was used to generate the specific anti-UL54 antibody. The UL54 protein was initially diffusely distributed throughout the cytoplasmic region; then, after 2 h, it gradually distributed into the nucleus, peaking at 24 h, and complete localization to the nucleus was observed thereafter. The UL54 transcript was detected as early as 0.5 h, and peak expression was observed at 24 h. The UL54 gene was insensitive to the DNA polymerase inhibitor Ganciclovir (GCV) and the protein synthesis inhibitor Cycloheximide (CHX), both of which confirmed that UL54 was an immediate early gene.The DEV UL54 gene was expressed in a prokaryotic expression system and characterized for expression level, intracellular localization and gene kinetic class. We propose that these results will provide the foundation for further functional analyses of this gene.

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