Preparation Of Polyclonal Antibodies Against Rotavirus Non-structural Protein,NSP2,and Study Of Its Interaction With NSP5 And Host Proteins

Objective To prepare polyclonal antibodies against rotavirus(RV)non-structural proteins NSP2,explore the interaction mechanism between NSP2 and NSP5,find out interaction factors between RV and host cells,and lay the foundation for in-depth exploration of the role of NSP2 in RV replication.Methods 1.Expression and purification of NSP2 recombinant protein: The extracted RV RNA was reverse-transcribed to synthesize c DNA,which was used as a template to amplify the fragments of NSP2,and then connected to the p GEX-6P-1 and p FLAG-CMV-3 vectors.The successfully constructed recombinant plasmid NSP2-p GEX-6P-1 was transformed into BL21(DE3)competent cells,and the optimal expression conditions were determined according to three aspects,IPTG concentration,temperature and induction time,so as to produce a large number of protein.High purity and concentration of NSP2 protein was obtained by GST affinity chromatography.2.Preparation of NSP2 polyclonal antibodies: The purified NSP2 recombinant proteins were respectively immunized BALB/c mice.The antisera were tested for titer and sensitivity by ELISA and Western Blot.3.Application of polyclonal antibodies: To verify the expressions of NSP2 in cells,Western Blot and IFA were uesd to detect MA104 cells infected with RV and 293 T cells transfected with NSP2-p FLAG-CMV-3 plasmid.4.Interaction between NSP2 and NSP5: The home-made NSP2 mouse polyclonal antibodies and NSP5 rabbit polyclonal antibodies were used to detect the RV-infected cells by laser confocal fluorescence to observe the localization of NSP2 and NSP5.To the end,Co-IP verfied the relationship of NSP2 and NSP5.5.Screening of host-cell proteins interacting with NSP2 through GST pull-down combined with protein profiling: target proteins,NSP2 purified protein and GST control protein,captured proteins in MA104 cells through GST pull-down.After silver staining,different bands were screened out for the corresponding proteins by protein profiling.Results 1.Expression and purification of NSP2 recombinant protein: The NSP2-p GEX-6P-1 recombinant plasmid was successfully constructed and then transformed into E.coli BL21(DE3)to induce protein by IPTG.SDS-PAGE showed the best induction conditions for NSP2 were 28 ℃,8 h and 0.4 mmol/L IPTG.The recombinant protein with high purity and concentration was obtained by GST affinity chromatography.2.Preparation of NSP2 polyclonal antibodies: BALB/c mice were immunized,and polyclonal antibodies successfully prepared.Western Blot showed they were very sensitive;ELISA demonstrated the titers of NSP2 polyclonal antibodies can reach 1:25,600.3.Application of polyclonal antibodies: NSP2 polyclonal antibodies can specifically detect cells infected with RV and transfected with NSP2-p FLAG-CMV-3 plasmid.4.Interaction between NSP2 and NSP5: Confocal laser microscopy showed that NSP2 and NSP5 were co-localized in infected cells,and Co-IP confirmed the interaction between the two proteins.5.Screening of host-cell proteins interacting with NSP2 through GST pull-down combined with protein profiling: SDS-PAGE and silver staining showed that NSP2 group and GST group had different bands in terms of capturing host proteins.Protein profiling screened out Pyruvate kinase,HSP90,RPS4,Ezrin and other proteins in MA104 cells,which can interact with NSP2.Conclusions 1.NSP2 recombinant plasmids were successfully constructed,and high-purity and high-concentration NSP2 recombinant protein was obtained.2.We successfully prepared high-sensitivity and high-titer NSP2 polyclonal antibodies.3.Prepared polyclonal antibodies can specifically recognize the expression of cells transfected with NSP2-p FLAG-CMV-3 plasmid and infected with RV.4.The interaction between NSP2 and NSP5 was observed.5.In MA104 host cells,many proteins interacting with NSP2 were successfully found,providing a basis for screening antiviral therapeutic targets.