Expression, Purification And Characterization Of NTPase/RNA Helicase Domain Of Dengue Virus Type 2 Nonstructural Protein 3 And Screening Its Antagonistic Peptide

Dengue viruses belong to the flaviviridae family and are transmitted via mosquitoes to millions of people each year in tropical and subtropical regions of the world. They consist of a group of four serologically related viruses referred to as dengue virus types 1- 4, with infection resulting in a range of clinical diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. There is currently nofever, dengue hemorrhagic fever, and dengue shock syndrome. There is currently no approved vaccine or effective antiviral therapy for these diseases. A comparison of the genomic sequences of four dengue virus serotypes has revealed that the NS3 protein is one of the most conserved viral proteins and shows the least sequence variation among the four serotypes of the dengue virus. The carboxyl-terminal region of NS3 encodes both nucleoside triphosphatase and helicase activities. All facts suggest that the NS3 protein play an important role in virual replication and maturation of infectious dengue virions. Our research work aims st constructing an expression system that could express bioactive NS3 protein and screening its small peptide ligands from which to single out its pioneer prohibitors. Generally, our work was conducted as follow:1) Construction of the expression system of DEN NS3 protein:DEN2 NS3 gene was cloned from DEN-2 infected cells by RT-PCR and inserted into the pET vector, then it was transformed into E.coli. The expression and purification condition was optimized. The recombinant protein was identified by SDS-PAGE and Western blot. DEN2 dNS3 was expressed with C-terminal histidine tag in E.coli and purified in a soluble form.2) Assay of NTPase activity and immunoreactivity of the recombinant dNS3A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green was used to measure the NTPase activity of DEN2 dNS3. The purified protein has NTPase activity and good antigenicity. We found that the NTPase activity of DEN2 dNS3 were stimulated by the DEN2 viral NS5 protein and polyA, which suggested that NTPase activity of NS3 protein may be regulated by the viral NS5 protein during virus replication. It is likely that the interactions between the NS3 and NS5 proteins may have induced a conformational change in the active site of NS3, which in turn enhances the substrate availability and hence NTP hydrolysis. This characteristic, together with the fact that NS3 is a multifunctional protein mediating a variety of diverse functions, has made it an interesting target for therapeutic intervention in flavivirus infections.3) Screening antagonistic peptide of dNS3 protein:...