| Dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) are vector-borne infectious diseases caused by dengue viruses (DENV). There are four antigenically related serotypes referred to as DENV-Ⅰ, DENV-Ⅱ, DENV-Ⅲ and DENV-Ⅳ. These diseases caused by Aedes aegypti and Aedes albopictus mosquitoes sting to people and epidemic in tropic and subtropic areas worldwide. It is estimated that50million dengue infections and500,000DHF cases occur each year. The most epidemic areas are mainly in south-east Asia, the western Pacific and the Americas. Currenlty, no effective antiviral drμgs and vaccines against dengue virus are for patients with DF and normal popμlation.The dengue viral genome consists of a single positive sense RNA of11kb and encods a single polypeptide chain. The signal peptide is dissolved into three structural proteins (C, M/prM and E) and seven nonstructural proteins (NS1, NS2A/B, NS3, NS4A/B, NS5) in the role of host signal peptidase and viral protease enzyme (NS3). The NS3protein plays an important role in the flavivirus proteases encoding by its own poly-protein.The non-structure3(NS3) protein of DENV is the second largest non-structure protein, which the length is1854bp and encode618amino acids. The NS3protein is a hydrophilic mμltifunctional protein and has the activity of protease, RNA helicase and RNA polymerase. These activities play an important role in the replication and maturation of DENV. In the process of formatting the mature virions, the protein C precursor is cutted by the NS2B/NS3protease and the prM and E proteins’side chains are properly processed by the host cell glycosyltransferase. NS2B/NS3catalyzed protein C in the carboxy-terminal cytoplasmic side in the ER lumen, so that the prM signal peptide can do Brownian motion in the process of translocation channel. Then, the prM signal peptidase is recognized and further processed in the luminal side of the ER membrane. Hence, the NS3protein has been the target of against dengue virus.NS3protein located in the endoplasmic reticμlum is an outer membrane protein. It contains two domains. One is a serine protease domain within the first180amino acids at the N-terminal portion, while the1-167residues seems to be the minimum sequence required for the proteolytic activity in DENVII. The other contains three enzymatic domains:RNA stimμlated nucleoside triphosphatase (NTPase), RNA helicase and RNA59-triphosphatase (RTPase), which participate in the virual precursor, virus replication and5’caping of dengue virus genome. All of above functions are to adjust the topology of nucleic acids.Other wise, Helicase may also disrupt the formation of secondary structures or substitute other virus host protein binding to other dsRNA. It is easier for viral RNA replication. The association of RNA5’triphosphate activity and5’ capping reaction may exist, as the fist step of capping is hydrolyzing the5’ end phosphate. The activity of5’ triphosphate was confirmed by Bartehna et al. It is reported that NS3protein had good immunogenicity and specific CD4+and CD8+epitopes that can induce B, T cell responses. NS3protein faciniates the fusion of virus and cell membrance with the assistance of NS2B protease and followed by induced apoptosis.Currently, the new stragey of antivial drμgs is that the compounds target specific enzymes of virus for inhibing virus. This stragey has achived a large efficiency in the antivrial treatment of hepatitis virus C (HCV) which is also belonged to the flavivirus. The drμgs of NS3inhibitors have been in the clinical trials and the antiviral outcome is efficiency. As NS3protein has the activity of proteases which is essencial in virus replication, excellent immunogenicity and conservative among the flaviviruses. According to that, the NS3of dengue virus has been protential antiviral target.To further investigate the role of NS3protein of dengue virus infection and the likelihood of antivial target, we construct the vectors of NS3protein in prokaryotic and eukaryotic by cloning, expression and purification. Thus, we can explore that the NS3protein regμlates replication of dengue virus; and we proved an important basis for further experiments to clarify the pathogenesis of dengue virus and a usefμl evidence for drμg design and vaccine against viral infections.The following is the main resμlts and conclusions in our research:1. The bioinformatic resμlts of NS3protein of dengue virus1.1The NS3domain of flaviviruses, such as DENV, JEV, TBEV, WNV and YFV, was analyzed by thebioinformatic softwores. NS3protein was homology in flaviviruses. The NS3protein was the closest relationship with other serotype of dengue viruses and closer with JEV and WNV, based on the evolutionary tree.1.2The rare condons were existed in the NS3gene of DENV-II by the analysis of Rare Codon Calcμlator. The NS3protein was located outside of the membrane protein and no transmembrane domain and no N-terminal-glycosylation sites were existed by the prediction of TMHMM. It is located in the endoplasmic reticμlum of the host and viral capsid by the analsis of Virus-mPLoc.2. Prokaryotic expression, purification and immunogenicity of DENV-II NS3protein2.1Prokaryotic expression and affinity of recombinant NS3proteinsThe RNA of dengue virus was extracted from neonatal rat brain, specific primers were designed for amplifing DENV-II-NS3gene. The fμll-length NS3gene was connected into pMD18-T vector, followed by screening clones, restriction enzyme digestion and DNA sequencing. Then we got the new recombination plasmid (PMD18-T-DENV-II-NS3). Using this plasmid as a template, we amplied the NS3gene with other restriction enzymes sits, instered the gene into the vector of pET32a, Transformed into E. coli BL21(DE3) and achived the Pet32a-NS3/BL21(DE3) clone. This clone was induced and lysised by IPTG and sonication. The expression products were got. The products conatined NS3protein and the protein existed as inclusion body. After the Ni-NTA affinity purification, the specific protein band which is marked the size of NS3protein was achieved. The Western blot confirmed the protein was the desired protein, so this also sμggested that the NS3protein has immunogenicity. The purified NS3protein is for the immunization antisera prepration. It provided a good tool for the further mechanism study of the role in DENV-Ⅱ infecting host cell.2.2Biological function of NS3proteinAfter extracting plasmid DNA from the mass of plasmid pcDNA-NS3P, pcDNA-NS3H, pcDNA-NS3and pcDNA (+), using lip2000transiently transfected BHK-21cells and attacking BHK-21cells by DENV-Ⅱ with titers of MOI=1. The total RNA of the cells was collected after72h and the viral load was determined by qPCR. The resμlts showed that the differences were existed between the group of transfection pcNS3, pcNS3H group and control and non-transfected group (p<0.05). No singinficantly difference was existed in the control and non-transfected group (p>0.05). Althoμgh the group of tranfection with pcNS3P group existed variation of virus titers compared with control and non-transfectd group, the variation was no significant difference (p>0.05). Therefore, DENV-Ⅱ-NS3protein has the effect in DENV-Ⅱ replicaiton in vitro.Using prokaryotic and eukaryotic expression vectors for constructing recombination NS3plasmid, this study confirmed DENV-Ⅱ non-structural protein3(NS3) played a role in inhorbing dengue replication. |
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