Preparation Of Ebola GP Protein Antigen And Screening Of Monoclonal Antibody

Ebolavirus(EBOV)belongs to the filamentous virus family and the viral nucleic acid is a single strand of negative strand RNA.EBOV-induced hemorrhagic fever was first reported in 1976 with a lethality of 90%.In 2014,West Africa broke out in the history of the most serious Ebola epidemic,resulting in more than 20,000 people infected,18,000 deaths.Therefore,the development of a preventive vaccine with good immunogenicity has become the primary task of preventing the spread of Ebola.Studies have shown that the infection process of EBOV is closely related to the type I transmembrane protein GP encoded by the viral gene,and GP is the only surface envelope protein of EBOV,so GP is the most ideal antigen to induce anti-EBOV neutralizing antibody.In the study of EBOV for the prevention and treatment of drugs in the drug because of relatively safe,targeted specific,small side effects become a hot spot.2014 antibody drugs ZMapp achieved remarkable curative effect.ZMapp monoclonal antibody is mixed injection,the main ingredient of MB-003(also known as: Mapp)and ZMAb.MB-003 is composed of three against ebola monoclonal antibody,including two single resistance strains is the source of the rat.Therefore,to develop the source of the rat monoclonal antibody in EBOV diagnosis and treatment has a realistic significance.Studies have shown that the recombinant protein vaccine can be induced inclusive neutralizing antibody,at the same time,because of the advantages on transportation,use,recombinant protein vaccine suitable for poor health condition of the region.Recombinant protein vaccine development is the bottleneck of GP protein in a short time,a lot of preparation.Protein expression system includes prokaryotic and eukaryotic expression system.In the eukaryotic expression system,HEK293 cell line and CHO cell line are the two most commonly used cell lines.In particular,HEK293 cells transient expression system,because no need to filter cell lines,can be prepared in a relatively short period of time a large number of recombinant proteins,in the prevention and control of infectious diseases widely used.Therefore,the eukaryotic expression plasmid pXG4678-mod-GP-Fc-2 was constructed and the recombinant protein of Ebola virus GP was obtained by transiently transfected HEK293 F cell platform.The recombinant eukaryoticexpression system was analyzed by analyzing the eukaryotic expression system.The factors influencing the expression of recombinant protein were determined by optimizing the optimal conditions of transient transfection,thus greatly improving the expression level of GP recombinant protein and establishing a HEK293 F transient transfection technique platform.In this study,BBC cells and mouse myeloma SP2 / 0cells were immunized with the recombinant protein GP expressed by the above method using hybridoma cell technology.The Ebola monoclonal antibody was prepared and detected and identified.The results showed that the recombinant protein of eukaryotic expression plasmid constructed in this study was consistent with the Ebola virus surface glycoprotein GP in biological structure and function,and could be used for biological detection.The successful establishment of the suspended cell HEK293 F The transfection technique platform could increase the expression level of GP recombinant protein by more than 7 times,lay the foundation for the subsequent antibody study.Four monoclonal antibodies were successfully screened by hybridoma technique,and all four antibodies were specific to GP protein Antibodies,although not neutralized but have good binding activity,lay the foundation for the treatment of antibodies against Ebswald.