The Effect Of Aptamers Against Hepatitis C Virus Nonstructure Protein NS2on Virus Lifecycle

Hepatitis C virus (HCV) infects170million people worldwide and has emerged as amajor causative agent of serious liver disease, such as chronic hepatitis, cirrhosis and evenhepatocellular carcinoma. A protective vaccine is not yet available and therapeutic options arestill limited, so it is urgent to develop more effective and better tolerated therapies for chronichepatitis C. For the role that HCV nonstructural protein NS2can affect assembly of progenyvirions and interfere with the physiological processes of the host cell to facilitate virusreplication, it has high potential to act as a target for developing antiviral drugs. Aptamer is anew type of recognition molecules which has a strong affinity, high selectivity, good stability,easy modification and other advantages. Aptamer is widely used in biological monitoring anddrug development. Currently there is no report about the drugs targeting NS2protein. In thisstudy we screened the functional ssDNA aptamers which can specifically bound to NS2protein using SELEX technique. We also examined the effect of these aptamers on HCVlifecycle and the following results were obtained。(1) The full-length NS2gene of HCV was successfully amplified by Polymerase chainreaction (PCR) using pJFH1as a template and the targeted gene was cloned into theprokaryotic expression vector pET28b.(2) The recombinant plasmid pET28b-NS2was transferred to BL21competent cells.Lauric acid, a membrane surface active reagent, was added to enhance the solubility of thefusion protein expressed in E.coli and the histidine-tagged protein was purified by Ni-NTAagarose.(3) After5rounds of seletion, we selected three ssDNA aptamers with high-affinity toNS2protein (namely NS2-1, NS2-2and NS2-3). These ssDNA aptamers can bind specificallyto NS2protein other than control protein such as NS5B and LacZ protein.(4) We used RT-PCR and immunofluoresence to detect virus replication and releasingafter transfected the aptamers to cells. The results showed that these aptamers can inhibitHCV RNA replication in a dose-dependent manner. For the more, ssDNA aptamer NS2-2andNS2-3can also prevent production of infectious virus. So the effect of aptamers against HCVNS2protein can suppress HCV replication and assembly of progeny virions, it is expected tobe a new type of anti-HCV therapeutic drug.