Activity Assay And Inhibitor Screening Of HCV RDRP

Hepatitis.C.Virus(HCV),the major etiological agent of the chronic hepatitis.Presently,it is estimated that about 3% of the population worldwide has infected HCV. Which can develops into cirrhosis and hepatocellular carcinoma.The viral genome is a 9.6kb long positive polarity RNA molecule that comprises a single open reading frame,which encodes about 3010 amino acid polyprotein.This polyprotein precursor is subsequently processed by a combination of host cell and virally encoded proteases.This result in the generation of viral structural protein ,non structural protein.and a small membrance protein.As a RNA virus ,having a high mutation The development of the cell and animal models for HCV have equa;y slow and diffcult.Nowday,There are no effective vaccines produce.Current HCV therapies are based on INF-αor the combination of INF-αand ribavirin.But,this therapies can yield a sustained virologic response less than 50% of patients infected by HCV.In fact,the current therapy has significant side effects and expensive,novel and more efficacious therapies for HCV infection are urgently needed.NS5B is the RNA-dependent-RNA polymerase and is the catalytic core of the viral replicase.uninfected HCV cells normally don't expressNS5B.The biochemical characterization and crystal structure of NS5B have been reported by several groups.Regarded as an effective drug target some have been studied in a phaseⅢclinical study.We are interested in study on①Cloning of NS5B gene and construction of cloning plasmid.②Expression and purification of NS5B in Rosetta.③Establish a system of activity assay and select inhibitor from hypothesized screening.④Study mechanism of inhibitor and assay its IC50 value.1.Cloning of NS5B gene and construction of cloning plasmid.We first obtained the DNA sequence encoding NS5B by PCR using specific cloing primers.Then the purfied PCR production was inserted into pET-21a vector.It showed that the recombinat cloning vector was constructed after sequencing.2. Expression and purification of NS5B in RosettaThe constructed expression vectors were used to transform Rosetta cells.,grow at 37 until OD600 reached 0.6-0.8 and then were induced with 0.5mMIPTG..After growth at 30 for 5h cells were harvested. Harvested cells were resuspended in buffer and lysed in PH7.5.NS5B protein were purified using sequential chromatographic columns(Histrap ,SP-Sepharose column),Then analyse by SDS-PAGE,and about 1.2mg pure protein was obtained per litre of bacterial culture.3.Established a system of activity assay in vitro and select inhibitor from hypothesized screening.A typical polymerase assay for NS5B was performed using polyA as template ,oligodT as primer UTP andαP32-UTP(10μM) as substrate,purified NS5B 8μg/ml,PH7.5.React at room temperature for15min,and terminated by 100mMEDTA,then quantified by liquid scintillation counting.22 compound were selected by hypothesized screening dissolve in DMSO 3 compound showed a good inhibition for NS5B in 100μM.Number 24 is an important natural product,which is the major component of Chinese medicine.4. Study mechanism of inhibitor and assay its IC50 value.IC50 values were determined in NS5B catalyzed reactions that include 10μM UTP andαP32-UTP,purified 8μg/m NS5B,PH7.5.React at room temperature for 15min.Km value for UTP is determined from Michaelis-Menten equation. Under the small molecular concentration different condition,assay Km change along with substrate, 24 compound to NS5B possible be Counter- competitive inhibition.This research ,screens through the experiment,obtains the better inhibitory compound,One kind is the Chinese native medicine effective component, in domestic and foreign has not seen the correlation report. For HCV RDRP the NS5B protein character further research, inhibitor highflux screening and discussed 24 compound action mechanism from the molecularlevel and the anti- HCV medicine development haslaid the foundation.