Protein Expression And Antibody Preparation Of Nucleo Protein Of Ebola Virus And Marburg Virus

The Ebola virus(EBOV) is a unique pathogen exists in middle Africa, theinfection of EBOV can result in a severe viral hemorrhagic fever (Ebola hemorrhagicfever, EHF), EBOV and Marburg virus (MARV) both belong to Filoviridae. But thereis an exception, the Bundibugyo ebolavirus (BEBOV) doesn't infect human, which isfirst found in Philippines. As the rapid increasing of global import and export tradedevelopment and the potential threat of international bio-terrorist organization, EBOVhas become a global public health problem rather than the unique problem for Africa.EBOV is also considered to have severe adverse effects on the gorillas of Africa. Theinfection of African subtype EBOV to human can lead to a EHF with90%fatality ratesor much more, and there is no feasible methods for the prevention and treatment ofEHF. The apparent symptoms of EBOV infection is vascular damage, immunedeficiency and blood stasis and coagulation, which are caused by immunosuppressionand systemic inflammatory response. Eventually leading to the loss of functions ofvarious organs, further resulting in shock, this is to some extent similar with the septicshock.The objective of this study: expression of NP protein of EBOV and MARV withbaculovirus expression system in sf9cells, prokaryotic expression and purification ofNP protein of four EBOV subtypes and MARV, immune rats with the purified proteinsto obtain NP polyclonal antibody, screening the positive monoclonal strains of fusedmyeloma cell prepared previously by our lab to obtain anti-Zaire ebolavirus (ZEBOV)monoclonal antibody with the recombinant NP protein, the recombinant NP proteinwas expressed with baculovirus expression system in sf9cells previously in thisstudy. The acquiration of the monoclonal antibody is to establish an EBOV antigencapture ELISA method. The purpose of MARV NP protein and antibody production isto design a control for EBOV detection method, and the establishment of control canexclude the non-specific reaction and improve detection specificity.This study consists of two experiments:1. Expression of Zaire ebolavirus (ZEBOV) and MARV NP gene in the baculovirus expression system and identification of its reactogenicity2. Prokaryotic expression and purification of EBOV NP, production of anti-rat NPpolyclonal antibody and screening of monoclonal antibodiesConclusion: Successfully expressed the NP gene of the ZEBOV and MARV withbaculovirus expression system. Western blotting showed that recombinant EBOV NPand MARV NP reacted with their specific positive serum respectively, displayed noserological cross-reactions. Indirect immunofluorescence assay (IFA) detection of sf9cells infected with recombinant baculovirus showed that the NP of EBOV and MARVget a obvious expression, showing strong fluorescence, but control cells showed nospecific fluorescence. This study laid the foundation for EBOV and MARVepidemiological investigation and diagnostic kits development. We Successfullyexpressed recombinant NP proteins of the four subtypes of EBOV and MARV, therecombinant NP proteins were purified efficiently, the purity rate is higher than90%.Polyclonal antibodies against four subtypes of recombinant NP proteins and MARVrecombinant NP protein were obtained in rat serum, serum titers were as follows:anti-MARV type titer:1/102400, anti-ZEBOV type:1/25600, anti-Sudan ebolavirus(SEBOV) type:1/51200, anti-Bundibugyo ebolavirus (BEBOV) type:1/25600,anti-Cote d'Ivoire ebolavirus (CIEBOV) type:1/12800. Anti-ZEBOV NP monoclonalantibody cell strains were successfully obtained and showed no immune cross-reactionwith other subtypes.