Is China Building Africa?

In this article, the authors address the question “Is China Building Africa?” by examining the true nature of China’s infrastructure development projects in Africa, and how the different players involved interact with each other

Characterization of the duck enteritis virus UL55 protein

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Cloning, expression and characterization of gE protein of Duck plague virus

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Isolation and characterization of a novel alphanodavirus

<p>Abstract</p> <p>Background</p> <p><it>Nodaviridae </it>is a family of non-enveloped isometric viruses with bipartite positive-sense RNA genomes. The <it>Nodaviridae </it>family consists of two genera: alpha- and beta-nodavirus. Alphanodaviruses usually infect insect cells. Some commercially available insect cell lines have been latently infected by Alphanodaviruses.</p> <p>Results</p> <p>A non-enveloped small virus of approximately 30 nm in diameter was discovered co-existing with a recombinant <it>Helicoverpa armigera </it>single nucleopolyhedrovirus (<it>Hear</it>NPV) in Hz-AM1 cells. Genome sequencing and phylogenetic assays indicate that this novel virus belongs to the genus of alphanodavirus in the family <it>Nodaviridae </it>and was designated HzNV. HzNV possesses a RNA genome that contains two segments. RNA1 is 3038 nt long and encodes a 110 kDa viral protein termed protein A. The 1404 nt long RNA2 encodes a 44 kDa protein, which exhibits a high homology with coat protein precursors of other alphanodaviruses. HzNV virions were located in the cytoplasm, in association with cytoplasmic membrane structures. The host susceptibility test demonstrated that HzNV was able to infect various cell lines ranging from insect cells to mammalian cells. However, only Hz-AM1 appeared to be fully permissive for HzNV, as the mature viral coat protein essential for HzNV particle formation was limited to Hz-AM1 cells.</p> <p>Conclusion</p> <p>A novel alphanodavirus, which is 30 nm in diameter and with a limited host range, was discovered in Hz-AM1 cells.</p

Expressing gK gene of duck enteritis virus guided by bioinformatics and its applied prospect in diagnosis

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Bioinformatic predictions revealed that the expression of the full-length gK gene (<it>fgK</it>) 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 <it>fgK </it>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 <it>gK </it>gene by designing a novel truncated <it>gK </it>gene (<it>tgK</it>). 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.</p> <p>Conclusions</p> <p>In this work, the DEV-<it>tgK </it>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.</p

Expression and intracellular localization of duck enteritis virus pUL38 protein

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

Polyclonal antibody against the DPV UL46M protein can be a diagnostic candidate

<p>Abstract</p> <p>Background</p> <p>The duck plague virus (DPV) UL46 protein (VP11/12) is a 739-amino acid tegument protein encoded by the <it>UL46 </it>gene. We analyzed the amino acid sequence of UL46 using bioinformatics tools and defined the main antigenic domains to be between nucleotides 700-2,220 in the <it>UL46 </it>sequence. This region was designated UL46M. The DPV <it>UL46 </it>and <it>UL46M </it>genes were both expressed in <it>Escherichia coli </it>Rosetta (DE3) induced by isopropy1-β-<smcaps>D</smcaps>-thiogalactopyranoside (IPTG) following polymerase chain reaction (PCR) amplification and subcloning into the prokaryotic expression vector pET32a(+). The recombinant proteins were purified using a Ni-NTA spin column and used to generate the polyclonal antibody against UL46 and UL46M in New Zealand white rabbits. The titer was then tested using enzyme-linked immunosorbent assay (ELISA) and agar diffusion reaction, and the specificity was tested by western blot analysis. Subsequently, we established Dot-ELISA using the polyclonal antibody and applied it to DPV detection.</p> <p>Results</p> <p>In our study, the DPV UL46M fusion protein, with a relative molecular mass of 79 kDa, was expressed in <it>E. coli </it>Rosetta (DE3). Expression of the full <it>UL46 </it>gene failed, which was consistent with the results from the bioinformatic analysis. The expressed product was directly purified using Ni-NTA spin column to prepare the polyclonal antibody against UL46M. The titer of the anti-UL46M antisera was over 1:819,200 as determined by ELISA and 1:8 by agar diffusion reaction. Dot-ELISA was used to detect DPV using a 1:60 dilution of anti-UL46M IgG and a 1:5,000 dilution of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG.</p> <p>Conclusions</p> <p>The anti-UL46M polyclonal antibody reported here specifically identifies DPV, and therefore, it is a promising diagnostic tool for DPV detection in animals. UL46M and the anti-UL46M antibody can be used for further clinical examination and research of DPV.</p

Expression and characterization of recombinant VP19c protein and N-terminal from duck enteritis virus

<p>Abstract</p> <p>Background</p> <p>Previous studies have indicated that the VP19c protein and its homology play similar roles in capsid assembly of all <it>Alphaherpesvirus </it>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.</p> <p>Methods</p> <p>A recombinant VP19c (rVP19c) and N-terminal were expressed in <it>Escherichia coli </it>(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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Expression and characterization of UL16 gene from duck enteritis virus

<p>Abstract</p> <p>Background</p> <p>Previous studies have indicated that the UL16 protein and its homologs from herpesvirus were conserved and played similar roles in viral DNA packaging, virion assembly, budding, and egress. However, there was no report on the UL16 gene product of duck enteritis virus (DEV). In this study, we analyzed the amino acid sequence of UL16 using bioinformatics tools and expressed in <it>Escherichia coli </it>Rosetta (DE3) induced by isopropy1-β-D-thiogalactopyranoside (IPTG). The recombinant protein was produced, purified using a Ni-NTA column and used to generate the polyclonal antibody against UL16. The intracellular distribution of the DEV UL16 product was carried out using indirect immunofluorescence assay.</p> <p>Results</p> <p>In our study, UL16 gene of DEV was composed of 1089 nucleotides, which encoded 362 amino acids. Multiple sequence alignment suggested that the UL16 gene was highly conserved in herpesvirus family. The UL16 gene was cloned into a pET prokaryotic expression vector and transformed into <it>Escherichia coli </it>Rossetta (DE3) induced by IPTG. A 60kDa fusion protein band corresponding to the predicted size was produced on the SDS-PAGE, purified using a Ni-NTA column. Anti-UL16 polyclonal sera was prepared by immunizing rabbits, and reacted with a band in the IPTG induced cell lysates with an apparent molecular mass of 60 kDa. In vivo expression of the UL16 protein in DEV infected duck embryo fibroblast cells (DEFs) was localized mostly around perinuclear cytoplasmic area and in cytosol using indirect immunofluorescence assay.</p> <p>Conclusions</p> <p>The UL16 gene of DEV was successfully cloned, expressed and detected in DEV infected DEFs for the first time. The UL16 protein localized mostly around perinuclear cytoplasmic area and in cytosol in DEV infected DEFs. DEV UL16 shared high similarity with UL16 family members, indicating that DEV UL16 many has similar function with its homologs. All these results may provide some insight for further research about full characterizations and functions of the DEV UL16.</p