Expression Of The Bioactive NS5 Of Dengue Virus In Prokaryocytes And Screening Its Antagonistic Peptide

Dengue virus is one of the most important members of the flavivirus family, the diseases caused by which range from dengue fever, dengue hemorrage fever to dengue shock syndrome. It has put populations of more than 100 countries or areas at risk. By now, there is neither reported effective substance nor certified vaccine. The NS5 protein of dengue virus is the largest molecule encoded by the virus genome with a molecule weight of 104 000. Recent research work indicates that the NS5 protein acts as the RNA-dependent RNA polymerase(RdRP) in virus which has the ability to recognize and bind its template RNA to synthesize a complementary strand. All facts suggest that the NS5 protein plays an important role in viral RNA replication, so it maybe a target to prohibit the viral replicating. By far, there is no such reported research work as screening its prohibitors or ligands. Our research work aims at constructing an expression system that could express bioactive NS5 protein and screening its small peptide ligands from which to single out its pioneerprohibitor. Generally, our work was conducted as follow:(1) Construction of the expression system of DEN NS5 proteinThe total RNA was distilled from tissues infected by dengue viruses. Then the NS5 gene was obtained by way of RT-PCR and was inserted into plasmid pQE30, followed by transforming into E coll XL 1-Blue. After it is confirmed that XL 1-Blue may express the NS5 protein, incubating conditions as culture and temperature during inducing expression period were optimized. Thus, we obtained the recombinant NS5 protein in form of dissolving. Purification was conducted with Ni-NTA resin as there's a 6 X His tag at the N-terminal of the recombinant protein. After stepwise washing and elution at purification procedure, sublimated protein was obtained with the full-length NS5 accounting for more than 90% and the aborted translated protein about 6.5%. We think this part of research is the basis of the whole task.(2) Assay of RdRP activity and immunoreactivity of therecombinant NS5 First we respectively cloned the 5' and 3' terminalregion(TR) sequence of the viral genome by RT-PCR and jointed the two segment by ligation. After transcription, we gained the subgenomic RNA that may play the role of the template RNA recognized by DEN RdRP. Secondly, we established a RdRP reaction system. Then the recombinant NS5 protein was added to the system and its RdRP activity was confirmed after the newly synthesized RNA showed resistant to RNase A. Furthermore, we found that the recombinant NS5 could specifically bind antibodies in anti-DEN serum indicating the immunoreactivity of the recombinant NS5 and the irnmunogenicity of DEN NS5. So, the recombinant NS5 protein maybe used in diagnosis of DEN-infectious diseases. On account of vaccination purpose, the neutralizing protection of anti-NS5 antibodies should be tested.(3) Screening antagonistic peptide of NS5 protein A library ofphage-displayed disulfide-constrained random peptides was incubated with a plate coated with the recombinant NS5 protein. Then the unbound phage was washed away, and the specifically-bound phage was eluted and amplified. After 4 rounds of panning, we selected 20 amplified phage clone to sequence their displayed peptides. 5 of them could not be workout. The other 15 peptides were classified into 2 groups, each group sharing a different consensus sequence that might bind a different site of the target. One peptide of each group was chemically synthesized and added to the RdRP reaction system. We found that one peptide refrained the activity of RdRP. After added to culture of DEN-infected cells, the peptide showed distinct protection to cells.