Construction Of SFTSV Preudovirus And Preparation Of Neutralizing Antibody

BackgroundSevere fever with thrombocytopenia syndrome(SFTS),commonly known as tickosis,is a zoonotic infectious disease caused by a novel Bunya virus(SFTSV).With the characteristics of rapid onset,high fatality rate and wide geographical distribution of the disease,it has posed a serious threat to public safety and global human health.SFSTV belongs to the genus Phlebovirus of the Bunyaviridae family.Its genome is divided into three fragments,L,M and S,which encode RNA dependent RNA polymemse(Rd RP)and glycoprotein precursor(GPC),nucleoprotein(NP)and nonstructural proteins(NSs).GPC precursor is translated and cut by proteases in host cells to form N-terminal proteins N(Gn)and C-terminal proteins C(Gc),which are mainly responsible for viral ligand binding with host cell surface receptors and membrane fusion,and contain the main epitopes of induction and neutralization antibody.It is a potential target for the development of SFTSV vaccines or therapeutic antibodies.As a virulent virus,SFTSV’s high pathogenicity and infectivity hinder its research.In order to improve the detection efficiency of SFTSV,we designed the pseudovirus detection system which relies on the carrier Vesicular stomatitis virus(VSV).As the model virus species in the construction of pseudovirus system,VSV has been widely used in the study of virus particle invasion of host cells,identification of cell surface receptors mediating virus infection of cells,screening of virus suppressor drugs,and vaccine development.Due to its extensive tolerance,we replaced the envelope G protein of VSV with the envelope Gn/Gc protein of SFTSV and packaged it into recombinant VSV(rVSV)pseudovirus.Reporter gene GFP/NLuc was introduced to facilitate qualitative and quantitative analysis.The pathogenesis of SFTSV is complex and diverse,so far there is no specific treatment and specific drugs to combat its infection.Symptomatic treatment is the main solution,and cannot eliminate the pathogen.Therefore,this study attempted to screen neutralizing antibodies against SFTSV M(JS2011,Jiangsu strain 2011).Firstly,SFTSV Gn-Fc recombinant plasmid was constructed based on eukaryotic protein expression vector pAB,and SFTSV Gn-Fc protein was expressed by 293 F cell transient transfection system and extracted and purified.Then,mice were immunized with Gn-Fc as antigen.monoclonal Antibody(mAb)for SFTSV Gn was prepared by hybridoma cell fusion technique.Then,the specific monoclonal antibody was screened using VSV-based SFTSV M pseudovirus successfully packaged in the previous stage and its function was evaluated.Finally,it was found that the prepared mAb had good reactivity and specificity,and had certain neutralizing activity through a variety of detection methods.In conclusion,this study focuses on SFTSV pseudovirus detection system and preparation and functional evaluation of neutralizing antibody,providing a good idea for the development of SFTSV diagnosis and treatment.Objective1.Utilize the optimized VSV reverse genetic operating system to "rescue" single-round and multi-round replicating rVSV-SFTSV M(JS)-GFP/NLuc pseudoviruses.2.By constructing the pAB-SFTSV Gn(JS)-Fc recombinant plasmid,293 F cells were transiently transfected with a high-efficiency expression protein system to express SFTSV Gn-Fc protein and extracted and purified as antigens.3.Hybridoma cell lines with antibodies against SFTSV Gn(JS)single epitopes were screened by immunizing mice,in vitro cell fusion and positive cell subcloning.mAb-SFTSV Gn(JS)was enriched by induced ascites in mice.4.Functional evaluation of monoclonal antibodies using Western blot,BLI,IFA,rVSV-SFTSV M(JS)pseudovirus reporting system.Methods1."Rescue" single-and multi-round replicating rVSV-SFTSV M(JS)pseudoviruses through the VSV reverse genetic system.(1)Single-round replicating virus: by constructing a pCAG-SFTSV M(JS)recombinant plasmid transfected into HEK293 T,and then infected with VSV-△G GFP virus,the expression of GFP was observed by inverted fluorescence microscopy,and the expression level of viral protein was detected by Western blot.(2)Multi-round replication virus: First,the rVSV-SFTSV M(JS)-GFP/NLuc recombinant plasmid was constructed by VSV virus vector to verify its expression.Then,the rVSV-SFTSV M(JS)-GFP/NLuc recombinant plasmid,E3 L expression plasmid and auxiliary plasmid(N,P,L,G,T7)were co-transfected into HEK293T:Vero E6(5:1)mixed cells,and the expression of GFP was observed by inverted fluorescence microscopy,NLuc luciferase activity was detected by multifunctional microplate reader,and the expression level of viral protein was detected by Western blot.2.Monoclonal antibody preparation: The recombinant plasmid pAB-SFTSV Gn carrying human Fc and mouse Fc tags was constructed based on the protein high expression vector pAB by molecular cloning technology,so as to facilitate the subsequent packet plate detection.After that,the recombinant protein was expressed by the eukaryotic 293 F cell protein expression system,and the protein was purified by affinity chromatography.The recombinant protein was analyzed by western blot.The extracted and purified target protein was used as immunogen to immunize BALB/c female mice aged 6-8 weeks for multiple times,and then mouse serum titer was detected,cell fusion was performed,positive cells were subcloned for multiple times,and monoclonal strains that could secrete SFTSV Gn antibody statically,proliferate and grow well were screened out.Then the antibody subtype was identified and a large amount of antibody was prepared by inducing ascites in mice.High concentration of mAb was purified from ascites by Protein A/G affinity chromatography after dilution.2.Antigen preparation: pAB-SFTSV Gn recombinant plasmids carrying human Fc and mouse Fc tags were constructed based on eukaryotic expression vector pAB.The SFTSV Gn-Fc protein was expressed by the transient transfection of 293 F cells into a high-efficiency expression protein system and extracted and purified,and the purity of the proposed recombinant protein was analyzed by Western blot,Coomassie Brilliant Blue staining.3.Monoclonal antibody screening and preparation: The purified Gn-Fc(mouse)protein was used as the immunogen to immunize BALB/c female mice aged 6-8 weeks for multiple times,and then mouse serum titer was detected,cell fusion was performed,positive cells were subcloned for multiple times,and monoclonal cell lines that could secrete SFTSV Gn antibody statically,proliferate and grow well were screened.Subsequently,antibody subtypes were identified and mass amplified for stability analysis.After that,antibodies were prepared by inducing ascites in mice,and appropriate amount of hybridoma cells were injected into the abdominal cavity of mice to produce ascites.The collected ascites were diluted and high concentration of mAb was enriched by Protein A/G affinity column acid elution method.4.Antibody function evaluation: mAb function detection by Western blot,BLI,IFA and in vitro neutralization experiments.Results1.The VSV reverse genetic system was used to successfully "rescue" rVSV-SFTSV M(JS)single-round and multi-round replicating recombinant pseudoviruses.2.The pAB-SFTSV Gn recombinant plasmids carrying human Fc and mouse Fc tags were successfully constructed,and the purified proteins were expressed by eukaryotic 293 F cell protein expression system with protein concentrations of 0.8 mg/m L and 1.3 mg/m L,respectively,and the protein purity and immunogenicity were good.3.Successful cell fusion,high fusion efficiency,the positive rate of initial screening reached 98.5%,and after the second subcloning reached 100%.4.Three SFTSV M mAbs were successfully screened,named mAb-1G12,mAb-8A1 and mAb-9A12.Both can specifically label and bind viruses,and mAb-1G12 and mAb-8A1 have neutralizing effects on viruses to a certain extent.ConclusionThis project successfully packaged SFTSV M(JS)recombinant pseudovirus through VSV optimized reverse genetics,and was applied in it and in the process of antibody development.The recombinant SFTSV M(JS)protein was successfully expressed by 293 F cell protein expression system,and three specific antibodies against SFTSV M were screened by hybridoma cell fusion screening technique,which were named mAb-1G12,8A1,9A12,respectively.All three antibodies showed good reactivity to ELISA,Western blot and IFA,and had certain neutralizing activity to mAb-1G12 and 8A1.This study provides clues for further research on the pathogenesis of SFTSV,and also provides a basis for the treatment of SFTS as well as the study of targeted drugs and vaccines.