This study aims to screen tags that enhance the soluble expression of pseudorabies virus(PRV)gD protein and to detect the biological activity of the resulting fusion proteins.Recombinant plasmids pET21b-MBP-gD,pET21b-SUMO-gD,pET21b-NusA-gD,and pET21b-GST-gD were constructed by PCR amplification of the PRV gD gene and linkage with four prokaryotic expression vectors,each carrying a different solubilization tag:MBP,SUMO,NusA,and GST.These plasmids were transformed into E.coli BL21(DE3).After induction,SDS-PAGE was used to detect the expression of each recombinant protein,and the tag yielding the highest soluble expression was identified.The results demonstrated that the MBP-tagged gD protein had the most significant soluble expression,followed by the GST-tagged protein.In contrast,the SUMO-and NusA-tagged proteins were expressed as insoluble inclusion bodies.Consequently,the rMBP-gD fusion protein was selected for further characterization.The rMBP-gD protein was purified using a Ni-NTA column,and its immunoreactivity was assessed by western blot and indirect ELISA.Indirect immunofluorescence assay(IFA)was used to evaluate its binding to PK15 cells.The CCK-8 assay was employed to determine the safe concentration of rMBP-gD on PK15 cells.Furthermore,PK15 cells were co-incubated with PRV-GFP,and flow cytometry was used to assess the competitive binding of rMBP-gD to PK15 cells and its potential to inhibit PRV infection.Western blot and indirect ELISA results confirmed the immunoreactivity of rMBP-gD.IFA results indicated that rMBP-gD could bind to PK15 cells.Flow cytometry results demonstrated that rMBP-gD competitively bound to PK15 cells,thereby inhibiting PRV infection.These findings indicate that the prokaryotically expressed rMBP-gD fusion protein retains biological activity.In conclusion,this study screened four different solubilization tags,successfully expressed a soluble PRV gD fusion protein,and preliminarily identified its biological activity.This work lays a foundation for further research on PRV genetically engineered subunit vaccines.
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