Preparation And Identification Of Single-chain Variable Fragment Antibody Against Ns1' And E Protein Of Japanese Encephalitis Virus

Japanese encephalitis is a mosquito-borne disease in Asia caused by Japanese encephalitis virus.JEV can not only infect waterfowl,pigs and mosquitoes,but also humans.For decades,traditional monoclonal antibodies have been used in the study of a variety of diseases,in addition,single-chain variable fragment antibody is increasingly used in the laboratory.Single-chain variable fragment antibody shows many advantages:firstly,it has small molecular weight,and it can reach the target tissue effectively;Secondly,it does not have a constant region of Fc fragments,so it will not bind to Fc receptors on non-target cells;Besides,the expression forms are diverse,including prokaryotic expression system,eukaryotic expression system,plant expression system.In view of this,this study intends to use Escherichia coli expression system to develop JEV NS1' single chain variable fragment antibody and apply it to the study of NS 1' protein,so that we can provide a new scheme for the study of Japanese encephalitis virus.Compared to E.coli expression system,Bac-to-Bac baculovirus expression system is a good choice for preparation of single-chain variable fragment antibody because it can preserve natural structure of proteins.So this study expressed a kind of flavivirus-pan reactivity E protein single-chain variable fragment antibody in sf9 cells,and these two single-chain variable fragment antibodies expressed in different expression system were compared,specific as follows:1.Prokaryotic expression and identification of single-chain variable fragment antibody against NS1' protein of JEVHeavy and light chain variable regions gene of the JEV NS1' monoclonal antibody were amplified from a murine hybridoma cell strain respectively,and single-chain variable fragment antibody gene was obtained using overlap extension PCR(SOE-PCR).Then the single-chain variable fragment antibody was expressed in Escherichia coli,and expressed products were purified by Ni-column affinity chromatography after ultrasonic decomposition.Results show as follows:The relative molecular mass of JEV NS1' single-chain variable fragment antibody was 33 kDa,it could recognize specifically with HRP conjunct anti-His tag mouse monoclonal antibody.Confocal microscopy analysis showed that the single-chain variable fragment antibody was able to recognize JEV antigen in cells.Therefore,JEV NS1 'single-chain variable fragment antibody was successfully prepared,providing an experimental basis for the screening of recombinant antibodies against Japanese encephalitis virus derived from a single B cell.2.Expression and identification of single-chain variable fragment antibody against E protein of JEV in baculovirus expression systemMonoclonal antibody 6B6C-1 has pan-flavivirus cross reactivity,its variable region gene sequences of heavy chain and light chain have been published in NCBI(FJ234928.1,FJ234927.1).According to these foundations,the single-chain variable fragment antibody gene of 6B6C-1 containing His label was synthesized,and this gene was cloned into pFastBacDual,then recombinant bacmid was obtained by transforming recombinant vector pFastBacDual-scFv-E to the Escherichia coli competent cell DH1OBac.Then Spodoptera frugiperda ovarian cell line Sf9 was transfected with recombinant bacmid.72 h later,the transfected Sf9 cells were centrifuged and supernatant was collected to obtain infectious recombinant virus,which was used to infect newly cultured Sf9 cells.After three generations of culture,cells were collected,and protein was purified by Ni-column affinity chromatography after ultrasonic decomposition.The purified single-chain variable fragment antibody was identified by coomassie blue staining and Western-blotting.Results showed that:pFastBacDual-scFv-E recombinant vector was successfully constructed.After transfection of recombinant bacmid into sf9 cells,these cells could express single-chain variable fragment antibody of E protein,and all of P1,P2,P3 baculoviruses had infectious ability.The purified single-chain variable fragment antibody was of high purity with a concentration of 0.1 mg/mL.Western-blotting results showed that the antibody could specifically react with HRP-conjugated anti-mouse His labeled monoclonal antibody,and could specifically recognize the purified vaccine strain SA14-14-2 and the prME protein produced by transfected BHK-21 cells.In this study,6B6C-1 single-chain variable fragment antibody with binding activity of Japanese encephalitis virus was prepared,which laid a foundation for further investigation of its neutralizing activity in vitro and neutralizing JEV in the central nervous system so as to achieve the therapeutic effect of JE.