Establishing And Evaluation Of Antigen And Antibody ELISA Detection Method Of Marburg Virus

Marburg hemorrhagic fever(MHF)is a zoonotic infectious diseasecomposed of Marburg virus(MARV),which can causesevere hemorrhagic fever and whose mortality rate is almost 90%,there is not still drug commercialization.The virus for the first time in a laboratory of Marburg city in he Germanand was therefore named the most severe cases,which mainly occurred in bad medical equipment of Africa.Marburg hemorrhagic fever mainly spreads through close contact,therefore we should establisha rapidand safety detection method to control the spread of the disease.The genome of MARV is formed by single stranded RNA virus,genome is about 19.1kb,the viral genome contains 7 open reading frames(ORF),respectively,encoding 7 proteins,including nuclear protein(NP),envelope glycoprotein(GP),VP30 protein and VP35,VP40,VP24 protein and dependent RNA polymerase(L protein).The virus is has only a serotype.MARV glycoprotein(Glycoprotein,GP)is the only a protein on the surface of the virus,which can induce neutralizing antibodies,GP is comprised of GP1 and GP2,the receptor binding domain(RBD,aa38-188)is located in the GP1 N end,which can be better intact with cell receptor binding respond than GP1.We expressed the recombinant MARV-GP-RBD protein in Escherichia coli expression system,established indirect ELISA method based on the protein,prepared the monoclonal antibody and polyclonal antibody.In addition,We also established double antibody sandwich ELISA method for detection the glycoprotein content in Marburg virus like particles.Objective Receptor Binding Domain(RBD)protein of GP of Maburg Virus was expressed in prokaryotic cells and purified to produce the polyclonal antibody of rabbit against MARV-GP-RBD protein.Methods According to the genes equences of MARV GP published on Gen Bank,a pair of primers was designed,which was applied for amplifying the RBD of MARV GP by PCR method.Double digested PCR products were cloned into the prokaryotic expression vector p ET-30a(+)to generate the recombinant expression plasmid p ET-30a(+)-GP-RBD,which was transformed into E.coli BL21(DE3)for expressing MARV-GP-RBD proteins under variously inducing conditions(time,IPTG concentration,temperature).Recombinant protein was purified using His-Band Ni+affinity chromatography and purified recombinant protein was used to immunize rabbit and mouse to produce polyclonal antibody and monoclonal antibody.The immunogenicity and reactivity of the recombinant protein was identified by SDS-PAGE,Western-blot and IFA.Results A fragment about 453 bp in length of RBD gene was successfully amplified,recombinant plasmid p ET-30a(+)-GP-RBD was constructed correctly inserted into BL21(DE3)after PCR restriction analysis and sequencing.SDS-PAGE indentified a 25 ku protein was fully expressed as inclusion bodies with induction performed using 0.4 mmol/L IPTG at 37? for 7 h.After purification by His-Band Ni+,affinity chromatography,~20mg target protein was produced from 1L of shake flask culture which determined by BCA protein assay kit.Western blot confirmed that the recombinant protein could be recognized by monoclonal antibody against His,polyclonal antibody and monoclonal antibody,which showing good immunoreactivity;IFA test showed that polyclonalantibodie and monoclonal antibody could successfully identify recombinant baculovirus r Bacmid-GP-VP40 which expressing MARV GP,proving to have good immunogenicity.Conclusion Receptor Binding Domain(RBD)protein of GP of Maburg Virus was successfully expressed and purified.In addition,the preparation of polyclonal antibody and monoclonal antibody were completed,which laied the foundation for the research of subunit vaccine and establishment of detection method of antigen and antibody for MARV.Establishment and preliminary application of indirect ELISA for detectingtheantibody of MARV.Objective Indirect ELISA detection method of MARV antibodywas established,and the relevant conditions of the method were optimized.Methods The purified MARV glycoprotein receptor binding domain protein was theantigen,high horse serumwhich was immunized MARV virus like particles was thefirst antibody,negative horse serum anti-MARVwas the negative control,HRP labeledGoat anti horse Ig G as the second antibody,we also optimizated the reactionconditions and evaluated the specificity and sensitivity.Results The optimal massconcentration of antigen was 4ug/ml;the optimal dilution of serum was 1:640;theoptimal concentration of enzyme labeled two was 1:5000;indirect ELISA optimizedmethod can detect specificityantibody of MARV.The Ebola virus,West Nile virus,Venezuelan equine encephalitis,Rift Valley fever virus positive serum showed noreaction;The intra and inter batch variation coefficients were less than 10%.Conclusion This study successfully established the indirect ELISA MARV antibody in detecting method,which provided a simple and rapid method.Establishment and preliminary application of detection of Marburg virus glycoprotein double antibody sandwich ELISA quantitative detection method.Objective Quantitatively detect Marburg virus particle glycoprotein content.Methods MARV GP specific monoclonal antibody was the capture antibody,and purified polyclonal antibody Ig G against MARV GP was used as the detection antibody,and the antigen was the prokaryotic expression of MARV GP.The reaction conditions,specificity,repeatability and sensitivity were optimized and analyzed.Result The optimal dilution of capture antibody and detection antibody respectively was 1:2560 and 1:8000;There was no cross reaction With the Ebola virus,West Nile virus,Middle East respiratory syndrome virus,Rift Valley fever virus.Intra and inter assay coefficients of variation were less than 10%,the detection sensitivity of MARV was 21.052 ng/ml.Conclusion This method could specifically detect MARV and did not intact with the Ebola virus,West Nile virus,Middle East respiratory syndrome virus,Rift Valley fever virus.There was great specificity,good repeatability,high stability,in detecting MARV GP.This method is convenient and accurate,which could monitor the relative content of glycoprotein in the Marburg virus particles.In this study,an effective,convenient and fast method for detecting MARV double antibody sandwich ELISA was established,whichthas good application value in vaccine production.