Purification Of Non-structural Protein (NSs) Of SFTSV And Preparation Its Polyclonal Antibody

ObjectivePreparation of a polyclonal antibody against the non-structural protein(NSs)of severe fever with thrombocytopenia syndrome virus(SFTSV),for further discovering the structure and function of the NSs gene,laying the foundation for further exploration of the pathogenesis of SFTSVMethods1.Expression and purification of PGEX-6P-SFTSV NSs recombinant protein:The SFTSV-NSs fragment was amplified by the cDNA synthesized by reverse transcription of the extracted RNA.The SFTSV-NSs fragment was amplified and ligated into the PGEX-6P-1 vector and the PFLAG-CMV-3 vector,respectively,to construct a recombinant plasmid.Construction of a successful recombinant plasmid PGEX-6P-SFTSV NSs was transformed into E.coli DH5?for cloning,and after identification,the correct recombinant plasmid was transferred to E.coli BL21 to express the recombinant protein.Following optimization conditions,the optimal conditions were selected,and the recombinant protein was induced in a large amount and filtered by GST affinity column chromatography to obtain high purity PGEX-6P-SFTSV NSs recombinant protein.2.Preparation of PGEX-6P-SFTSV NSs polyclonal antibody:The purified PGEX-6P-SFTSV NSs recombinant protein was mixed with complete adjuvant to prepare antigen,immunized New Zealand white rabbit,and then immunized four times with incomplete adjuvant.The antibody was tested for specificity and potency by Western Blotting and ELISA.3.Application of polyclonal antibody:VERO cells infected with SFTSV virus and 293 cells transfected with SFTSV NSs-Flag plasmid were subjected to Western Blotting and indirect immunofluorescence(IFA)to verify the expression of SFTSV in cells and it's expression site;SFTSV virus infection was used to prepare VERO cells then the prepared antibody was used to detection of SFTSV virus titers.Results1.Expression and purification of PGEX-6P-SFTSV NSs recombinant protein:The PGEX-6P-SFTSV NSs and SFTSV NSs-Flag recombinant plasmids were successfully constructed.PGEX-6P-SFTSV NSs recombinant protein was expressed in E.coli BL21.After a series of conditions,the optimal induction condition was at 28?,IPTG was added at a final concentration of 1.0 mmol/L.The protein expression was the highest at 12 h,and it was mainly in the form of inclusion bodies in the sediment.A large amount of this recombinant protein was collected and subjected to GST affinity column chromatography to obtain a higher purity PGEX-6P-SFTSV NSs recombinant protein.2.Preparation of PGEX-6P-SFTSV NSs polyclonal antibody:Successfully immunized New Zealand white rabbits to prepare rabbit anti-SFTSV NSs polyclonal antibody.The obtained rabbit antiserum was detected by Western Blotting,and its specificity was good.The titer was 1:64,000 by ELISA.3.Application of polyclonal antibody:The obtained rabbit antiserum was detected by Western Blotting and the specific expression of the virus and eukaryotic plasmid SFTSV-NSs was obtained in the cells.NSs protein in the form of inclusion bodies in cells was observed by IFA.The presence of NSs protein.SFTSV virus titer is 4.67X10~9FFU/mL.Conclusions1.Successfully construct PGEX-6P-SFTSV NSs recombinant plasmid and obtain high purity PGEX-6P-SFTSV NSs recombinant protein.2.Successful preparation of specific and high titer rabbit anti-SFTSV NSs polyclonal antibody.3.The prepared polyclonal antibody can specifically recognize the expression of NSs protein in cells after eukaryotic plasmid SFTSV-NSs and SFTSV virus-infected cells,and can be applied to subsequent experiments.