Preparation Of Monoclonal Antibodies Against Japanese Encephalitis Virus And Antiviral Effect Of Vimentin-Fc Protein

Japanese encephalitis virus(JEV),being a member of the family Flaviviridae,is usually infected to the human body and animal through mosquitoes and then may cause acute viral encephalitic and neurologic disease.This disease occurs mainly in Asia and the Western Pacific region,and has become one of the most serious encephalitis diseases which threats to human.The incidence of sequelae caused by JE is high,and the sequelae can affect the perception,movement,language performance,psychological and etc of patients.Three glycosylation protein(PrM,E,and NS1)of Japanese encephalitis virus are the main immune protective antigen.PrM and E protein are the structure protein of Japanese encephalitis virus.There are many reports about their structure and vaccine research.NS1 protein is the non-structure protein of JEV,it is involved in the early stages of viral replication,and it may also be involved in virus assembly and release.NS 1 protein doesn’t have neutralization antigenic epitope.It can induce complement-dependent cytolytic reaction and induce body produce protective immune in the presence of non-neutralizing antibodies conditions.Large amounts of NS1 protein existed in JEV cell culture supernatant,cell surface and intracellular.NS1 protein is a non-structural proteins of JEV,the vaccinated animals do not produce antibodies against NS1,and there is more virus NS1 protein epitope may induce a plurality of reaction no specific meaning in the post antibodies.The prepared monoclonal antibody can provide basis and a powerful tool for further research of the characteristics and function of envelope protein and its antibody.Vimentin is a class III intermediate filament protein predominandy expressed in developing embryo and in cells of mesenchymal orgin in the adult.Is shown to play an important role in vital mechanical and biological functions such as embryo development and diferentiation,cell attachment and migration,apoptosis,inflammation and immunological reaction.Research has shown that the expression of vimentin in neural cells correlates to the cell tropism of JEV.So we constructed a fusion protein Vimentin-Fc to the effect of anti-JEV assay.This will play a positive role in clinical research.1.Preparation of Monoclonal Antibodies against serogroup-specific antigen Japanese encephalitis virus.Three monoclonal antibodies(McAb)against Japanese encephalitis virus(JEV)protein of S A14-14-2 strain were produced.The suckling mouse brain tissue used to subcutaneously immunize BALB/c mice in this study.Then splenocytes from the immunized mice were fused with SP2/0 myeloma cells.The suckling mouse brain tissue were coated as the detection origin and indirect ELISA was used to screen positive hybridoma clones.Limited dilution method was performed to subclone the positive clones.After three cycles of subcloning,three McAbs against the JEV were obtained,and designated as 1C5,3B11 and 3C4 respectively.The McAb 1C5,3B11 and 3C4 belonged to the subtype of IgGland the light chains of all antibodies were type.The antibody titer of ascites were 1:2×104,1:2×105 and 1:2×105 respectively.2.Expression and antigentic characterization of the nonstructural protein NS 1 of Japanese encephalitis virus.NA cell was infected with Japanese encephalitis virus.After the pathological changes were occurrence,supematant was collected to amplify the NS1 gene by RT-PCR and fused with His by constructing prokaryotic expression plasmid pET32a-NS 1 and transforming Rosetta.After induction by IPTG,high inclusion body expression of fusion protein pET-32a-NS1 was obtained and purified.SDS-PAGE analysis and Western blotting showed that the fusion protein was 60 KD and had immunological activity.Western blot showed that the antibodies 1C5 and 3B11 were specific to JEV NS1.Indirect immunofluorescence assay showed that both McAbs recognized JEV NS1 protein in JEV infected NA cells.McAbs reported here may provide valuable tools for further investigation of the structure and function of JEV protein.3.Expression of the fusion protein Vimentin-Fc and the effects of anti-JEVReceptor trap therapy,which uses the soluble viral receptors to block the attachment and internalization of virus,has been developed as a treatment for virus infection.In this study,we have constructed a pET-32a-Vimentin-Fc expression plasmid for the economical and scale-up production of Vimentin-Fc fusion protein in E.coil.The virus pull-down assay demonstrated the binding activity of the Vimentin-Fc to JEV.The analyses of quantitative real-time PCR assay revealed that Vimentin-Fc could block JEV infection in vitro.Our results indicated that the fusion protein of Vimentin-Fc could provide a new way to inhibit cell invasion by JEV for further clinical purpose.