牛病毒性腹泻病毒NS5A原核表达及多克隆抗体制备OA

[Objective]Prokaryotic expression of bovine viral diarrhea virus(BVDV)non-structural protein 5A(NS5A)was obtained to prepare a polyclonal antibody against the disease.[Methods]Physicochemical properties of BVDV NS5A were analyzed using bioinformatics software.The recombinant plasmid pET-28a-NS5A was constructed and transformed into competent E.coli BL21 cells.NS5A was induced for expression,followed by urea denaturation and purification via Ni2+-NTA affinity chromatography.The purified recombinant protein was emulsified with adjuvant to immunize BALB/c mice multiple times for generating polyclonal antibodies.Titer of the antibody was determined by indirect ELISA,and properties characterized by indirect immunofluorescence assay(IFA)and western blot.[Results]The hydrophilic NS5A lacked a signal peptide and transmembrane domains.The successfully constructed prokaryotic expression plasmid pET-28a-NS5A was transformed to obtain the recombinant NS5A with an approximate molecular weight of 58 kDa.After urea denaturation and Ni2+-NTA affinity chromatography,highly purified recombinant NS5A was used to prepare polyclonal antibodies with a titer exceeded 1∶409 600.High reactivity and specificity of the antibody was confirmed by western blot and IFA.[Conclusion]The successfully prepared polyclonal antibody was highly specific against NS5A.It made further research as well as development of clinical diagnostic means of BVDV possible.