Shunchuan Zhang

Sichuan Agricultural University, Hua-yang, Sichuan, China

Are you Shunchuan Zhang?

Claim your profile

Publications (16)20.56 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: MicroRNAs (miRNAs) are a group of short (~22 nt) noncoding RNAs that specifically regulate gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of ~70 nt, are processed into mature miRNAs by cellular RNases III. To date, thousands of miRNAs have been identified in different organisms. Several viruses have been reported to encode miRNAs. Here, we extended the analysis of miRNA-encoding potential to the Anatid herpesvirus 1 (AHV-1). Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs. We then compared the 12 mature miRNAs candidates with the all known miRNAs of the herpesvirus family. Interestingly, the "seed sequences" (nt 2 to 8) of 2 miRNAs were predicted to have the high conservation in position and/or sequence with the 2 miRNAs of Marek's disease virus type 1 (MDV-1). Additionally, we searched the targets from viral mRNAs. Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs and 2 miRNAs have the high conservation with the 2 miRNAs of MDV-1. The result suggested that AHV-1 and MDV-1 should have closed evolutionary relation, which provides a valuable evidence of classification of AHV-1. Additionally, seven viral gene targets were found, which suggested that AHV-1 miRNAs could affect its own gene expression.
    Virology Journal 05/2012; 9:93. · 2.09 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A recombinant UL55 protein (pUL55) of duck enteritis virus (DEV), produced in Escherichia coli, was tested for diagnostic applicability in an indirect enzyme-linked immunosorbent assay (1-ELISA) as a coating antigen. Serum dilutions of 1:6400 (0.025microg) are the maximum detection limits of the pUL55-ELISA, according to the determined cut-off value of 0.330. Antigenic cross-reactivity investigation in heterologous sera of ducks failed to provide evidence that other viruses of ducks could hamper the serodiagnosis of DEV, and the inhibition assay revealed that the specific binding of antigen and antibody can be inhibited by pUL55, both of which demonstrated a good specificity of the established pUL55-ELISA. This assay was further validated by comparison with a commercial 1-ELISA based on DEV (DEV-ELISA) and a neutralization test (NT). The results suggested that the sensitivity of pUL55-ELISA was almost as good as DEV-ELISA but was much higher than NT. The established pUL55-ELISA is a rapid, simple, sensitive, specific, and inexpensive serodiagnosis for detecting antibodies against DEV and has a potential to complement the traditional assays for serodiagnosis of DEV; it can be used as a diagnosis alternative candidate for serologic surveillance of DEV infection.
    Avian Diseases 12/2011; 55(4):626-32. · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. 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. 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. 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.
    Virology Journal 02/2011; 8:82. · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.
    Virology Journal 01/2011; 8:241. · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet. The newly identified duck enteritis virus UL55 gene was amplified and cloned into pMD18-T vector after digestion to generate a recombinant plasmid pMD18-T/UL55 for the establishment of qRT-PCR as standard DNA. The results of agarose gel electrophoresis and melting curve analysis demonstrated the primers we designed for qRT-PCR were specific and available. We used β-actin as a reference gene for normalization and established two standard curves based on pMD18-T/UL55 and pMD18-T/β-actin successfully. Based on that, the transcriptional analysis of DEV UL55 gene was performed, and the result suggested the expression of UL55 mRNA was at a low level from 0 to 8 h post-infection(p.i.), then accumulated quickly since 12 h p.i. and peaked at 36 h p.i., it can be detected till 60 h p.i.. Nucleic acid inhibition test was carried out for analyzing a temporal regulation condition of DEV UL55 gene, result revealed that it was sensitive to ganciclovir. Synthesis procedures of DEV UL55 gene can be inhibited by ganciclovir. The method we established in this paper can provide quantitative values reflecting the amounts of measured mRNA in samples. It's available for detection and quantification, also can be used in DEV diagnosis. The DEV UL55 gene was produced most abundantly during the late phase of replication in DEV-infected cells and the transcription of it depended on the synthesized DNA. DEV UL55 gene is a γ2 gene which occurs last and have a strict requirement for viral DNA synthesis.
    Virology Journal 01/2011; 8:266. · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Most of the previous research work had focused on the epidemiology and prevention of duck enteritis virus (DEV). Whilst with the development of protocols in molecular biology, nowadays more and more information about the genes of DEV was reported. But little information about DEV UL53 gene and glycoprotein K(gK) was known except our reported data. In our paper, the fluorescent quantitative real-time PCR(FQ-RT-PCR) assay and nucleic acid inhibition test were used to study the transcription characteristic of the DEV UL53 gene. Except detecting the mRNA of DEV UL53 gene, the product gK encoded by UL53 gene was detected through the expression kinetics of UL53 gene by the purified rabbit anti-UL53 protein polyclonal antibodies. Western-blotting and indirect immunofluorescence assays were used to detect gK. From the results of these experiments, the UL53 gene and gK were respectively identified as a late gene and a really late protein. On the other hand, the indirect immunofluorescence assay provided another information that the intracellular localization of DEV gK was mainly distributed in cytoplasm. By way of conclusions, we conceded that DEV UL53 gene is a really late gene, which is coincident with properties of UL53 homologs from other herpesvirus, such as ILTV(Infectious Laryngotracheitis virus) and HSV-1(Herpes simplex virus type 1). The properties of intracellular localization about gK protein provided a foundation for further functional analysis and further studies will be focused on constructing of the UL53 gene DEV mutant.
    Virology Journal 01/2011; 8:235. · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function. The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells. In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene.
    Virology Journal 01/2011; 8:256. · 2.09 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The analysis on codon usage bias of gI gene of duck enteritis virus (DEV) may provide a basis for understanding the evolution and molecular characteristic of DEV, and for selecting appropriate host expression systems to improve the expression of target gene. A comparative analysis of the codon usage bias of the DEV gI gene and 22 other refrence herpesviruses gI-like genes was performed, by using the CAI, CHIPS and CUSP programs of EMBOSS and the CodonW 1.4 program. The results showed that DEV gI gene was strong bias towards the synonymous codons with A and T at the third codon position, and was low codon usage bias gene, presumed lower expression. The ENc-plot revealed that G + C compositional constraint is the main factor that determines the codon usage bias in the herpesvirus gI-like genes. The phylogentic analysis suggested that DEV was evolutionarily closer to avian herpesviruses, condon usage pattern in the 23 herpesviruses gI-like genes was phylogenetically conserved, host species and gene function also had a certain influence to the codon usage bias. There were 26 codons showing distinct usage differences between DEV and Escherichia coli, 24 between DEV and yeast, 31 between DEV and Homo sapiens. Therefore the yeast and E. coli expression system may be suitable for the expression of DEV gI gene if some codons could be optimized.
    Biomedical Engineering and Informatics (BMEI), 2010 3rd International Conference on; 11/2010
  • [Show abstract] [Hide abstract]
    ABSTRACT: In the study, we calculated the codon usage bias in the newly identified UL38 gene of Duck Enteritis Virus (DEV) and performed a correlation analysis of codon bias of DEV UL38 gene and its host animal. There was a strong bias in the DEV UL38 gene towards the synonymous codons with A and T at the third codon position. The amino acids codon usage bias in the DEV UL38 gene was compared with other 15 reference herpesviruses, and the cluster analysis suggested that UL38 gene's type and function also contributed to the codon usage of it. In addition, there were 42 rare condons in the ORF of the DEV UL38, and 24 codons showing distinct usage differences between DEV and E. coli, 12 codons between DEV and Homo sapiens, 25 codons between DEV and yeast.
    Bioinformatics and Biomedical Engineering (iCBBE), 2010 4th International Conference on; 07/2010
  • Shunchuan Zhang, Anchun Cheng, Mingshu Wang, Jun Xiang
    [Show abstract] [Hide abstract]
    ABSTRACT: Duck enteritis virus (DEV) is an alphaherpesvirinae that causes an acute, contagious, and fatal disease. In this study, the DEV-ULS3 gene from Key Laboratory of Animal Disease and Human Health of Sichuan Province was cloned and sequenced. Nucleotide sequence analysis revealed a 1032 bp open reading frame (ORF) theoretically encoding a 343-amino acid polypeptide was homologous to UL53 gene of alphaherpesvirinaes but there was just a predicted 226aa peptide , which could be expressed facilely, by using GENSCAN. The DEV-ULS3 gene had a base composition of 270 adenine (26.2%), 206cytosine (20.0%), 232 guanine (22.5%) and 324 thymine (31.4%). Compared with the ULS3 gene of other alphaherpesvirinaes, the DEV-ULS3 gene had a close evolutionary relationship with members of the Mardivirus genus. Therefore, the phylogenetic tree of ULS3 might be used as a basis for DEV classification. In conclusion, all data will provide a basis for further functional analysis of the DEV-ULS3 gene.
    Bioinformatics and Biomedical Engineering (iCBBE), 2010 4th International Conference on; 07/2010
  • Source
    Shunchuan Zhang, Anchun Cheng, Mingshu Wang
    [Show abstract] [Hide abstract]
    ABSTRACT: Codon usage bias in UL53 gene of DEV was analyzed in this paper, the results indicated that it had strong bias towards to the synonymous codons with A or T at the third codon position. The codon usage bias of DEV UL53 gene and 26 reference herpesviruses were analyzed by EMBOSS program and RSCU. In addition, Phylogenetic tree indicated DEV was evolutionarily closer to CaHV-2, CaHV-3, MeHV-1. The ENC-GC<sub>3S</sub> plot revealed that the genetic heterogeneity in UL53 gene of herpesviruses was constrained by G+C content at the third codon position. Conclusions: The extent of codon usage bias in the DEV UL53 gene was highly correlated with the gene composition. Analysis of correspondence through the SAS statistics software showed the yeast expression system might be more suitable for the expression of DEV genes.
    Bioinformatics and Biomedical Engineering (iCBBE), 2010 4th International Conference on; 07/2010
  • [Show abstract] [Hide abstract]
    ABSTRACT: Glycoprotein K encoded by the DEV-UL53 gene is one of the DEV envelope glycoproteins. Like most members of the alphaherpesvirinae, the genes in the UL region of its genome are well conserved, which means that the major DEV envelope glycoprotein K possessing similar functions of envelope glycoprotein K of alphaherpesvirinae plays an essential role in viral replication and cell fusion. Glycoprotein K sequence analysis indicates that the protein possesses typical characteristics of membrane glycoproteins containing five transmembrane helix structures and an NH<sub>2</sub>-terminal signal peptide cleavage site, which provide some information for its expression. The precursor gK has five hydrophobic domains, which consists of 343 amino acids and exhibits a molecular mass of 38.12 kDa. Through combining the result of estimating potential antigenic epitopes and secondary structure presumption, five B cell dominant epitopes can be initially definited, which locate at 60-62, 144-155, 157-160, 210-214 and 293-294 amino acids. In conclusion, all the data and consequences will provide a basis for further functional study of the DEV-glycoprotein K and provide necessary data for the new type clinical diagnosis of DEV and the development of new DEV vaccine.
    Bioinformatics and Biomedical Engineering (iCBBE), 2010 4th International Conference on; 07/2010
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Duck viral enteritis, which is caused by duck enteritis virus (DEV), causes significant economic losses in domestic and wild waterfowls because of the high mortality and low egg production rates. With the purpose of eliminating this disease and decreasing economic loss in the commercial duck industry, researching on glycoprotein K (gK) of DEV may be a new kind of method for preventing and curing this disease. Because glycoproteins project from the virus envelope as spikes and are directly involved in the host immune system and elicitation of the host immune responses, and also play an important role in mediating infection of target cells, the entry into cell for free virus and the maturation or egress of virus. The gK is one of the major envelope glycoproteins of DEV. However, little information correlated with gK is known, such as antigenic and functional characterization. Bioinformatic predictions revealed that the expression of the full-length gK gene (fgK) in a prokaryotic system is difficult because of the presence of suboptimal exon and transmembrane domains at the C-terminal. In this study, we found that the fgK gene might not be expressed in a prokaryotic system in accordance with the bioinformatic predictions. Further, we successfully used bioinformatics tools to guide the prokaryotic expression of the gK gene by designing a novel truncated gK gene (tgK). These findings indicated that bioinformatics provides theoretical data for target gene expression and saves time for our research. The recombinant tgK protein (tgK) was expressed and purified by immobilized metal affinity chromatography (IMAC). Western blotting and indirect enzyme-linked immunosorbent assay (ELISA) showed that the tgK possessed antigenic characteristics similar to native DEV-gK. In this work, the DEV-tgK was expressed successfully in prokaryotic system for the first time, which will provide usefull information for prokaryotic expression of alphaherpesvirus gK homologs, and the recombinant truncated gK possessed antigenic characteristics similar to native DEV gK. Because of the good reactionogenicity, specificity and sensitivity, the purified tgK could be useful for developing a sensitive serum diagnostic kit to monitor DEV outbreaks.
    Virology Journal 01/2010; 7:168. · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Duck virus enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans, which has produced significant economic losses in domestic and wild waterfowl. With the purpose of decreasing economic losses in the commercial duck industry, studying the unknown glycoprotein K (gK) of DEV may be a new method for preferably preventing and curing this disease. So this is the first time to product and purify the rabbit anti-tgK polyclonal antibody. Through the western blot and ELISA assay, the truncated glycoprotein K (tgK) has good antigenicity, also the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines and the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer. Indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks.
    Virology Journal 01/2010; 7:241. · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Virology Journal 01/2010; 7:162. · 2.09 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Prediction of B cell epitopes of duck enteritis virus (DEV) VP19c protein encoded by UL38 gene and expression in prokaryotic system were described in the present study. The major antigenic portion of VP19c protein of DEV was predicted and corresponding gene was amplified from the extracted DNA and cloned. The recombinant expression construction(pPAL7-truncated UL38) was identified by the polymerase chain reaction, restriction enzyme and sequencing analysis. The profinity eXact affinity-tagged fusion truncated VP19c protein with a molecular mass of 35 kDa was expressed by IPTG induction and was purified to near homogeneity by extracting inclusion bodies(IBs) as judged by sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. The purified truncated VP19c protein was recognized by antibody to DEV in western blot analysis.
    01/2010;