Recombinant Expression Of The C-terminal 70 KDa Fragment Of Dengue Virus NS5 And Preliminary Study On Its Application

Dengue virus(DENV)infection in humans may cause self-limited Dengue fever(DF),or even clinically more severe Dengue hemorrhagic fever(DHF)and Dengue shock syndrome(DSS).It is a mosquito-borne infectious disease mainly in tropical and subtropical regions,and is becoming one of the fastest growing infectious diseases in recent years.People are relatively sensitive to first infection with whichever of the four serotypes and obtain the lasting immunity to the same serotype virus.But,secondary infection of different serotype virus is more likely to cause DHF/DSS because of the antibody dependent enhancement(ADE),leading to high morbidity and mortality.So far,there is no antiviral drug against DENV,thus rapid diagnosis and symptomatic treatment is particularly important after infection.Non-structural protein 5(NS5)of DENV is the longest and the most conserved protein amongst the Flaviviridae family,which plays a vital role in viral life cycle through replication of viral RNA,mostly because of its C-terminal 70 kDa fragment(NS5/C70)predicted as RNA dependent RNA polymerase(RdRp)structure.Herein,it might be a potential target for blocking or inhibiting viral replication.In this study,we firstly sequenced DENV-2 NS5 gene conserved in our lab,found eight mutations,and designed primers to do site-specific mutagenesis.Thus,we got the wild DENV-2 NS5 gene and successively constructed two expression plasmid vectors,pQE30-NS5 and pQE30-NS5/C70,then established the prokaryotic expression system of M15/pQE30-NS5/C70.Finally,we obtained soluble expression of the recombinant polypeptide segment of NS5/C70 and optimized the expression conditions.Further,the purified recombinant NS5/C70 was coated and reacted to serums of the four serotype patients by ELISA,with a group of uninfected normal serums used as negative control.The results showed that the recombinant NS5/C70 did not only show intense positive reactivity to DENV-2 infected serum at serums dilution of 1:500~1:16 000,but also positive or intense positive reactivity to the other three serotype serums.Therefore,the recombinant polypeptide can be used for rapid or auxiliary diagnosis of DENV infection.Meanwhile,it can be used for vaccine development without causing ADE,because it is the key part of the NS5.All in all,our work will be helpful for developing novel DENV vaccines and quick diagnostic reagents.