Polyclonal antibody preparation and immunogenicity analysis of the Nsp10 protein in porcine reproductive and respiratory syndrome virus

【Objective】 This study aimed to construct a prokaryotic expression system for the Nsp10 protein of porcine reproductive and respiratory syndrome virus （PRRSV）， prepare polyclonal antibodies， and evaluate their antigenicity， thereby providing a theoretical basis for establishment of serological diagnosis methods and vaccine research and development based on Nsp10 protein.【Method】 The hydrophilicity/hydrophobicity， antigenic epitopes， and tertiary structure of the Nsp10 protein were analyzed respectively using ExPASy， IEDB， and SWISS-MODEL. The recombinant plasmid pET-28a-Nsp10 was constructed and transformed into Escherichia coli BL21 （DE3） competent cells for induced expression. The induction temperature， IPTG concentration， and induction time were optimized. The recombinant Nsp10 protein （rNsp10） was purified using a His-Bind nickel ion affinity column， and the purification effects were assessed by SDS-PAGE and Western blotting. The purified rNsp10 was used to immunize C57BL/6J mice， and antibody titers and isotypes were determined by the methed of indirect ELISA. The relative expressions of immune response-related genes in splenocytes were measured using real-time fluorescence quantitative PCR， while antibody reactogenicity was evaluated by Western blotting and indirect immunofluorescence.【Result】 The Nsp10 protein was hydrophilic and contained 19 B-cell antigenic epitopes， suggesting its potential as a target antigen for polyclonal antibody preparation. The recombinant plasmid pET-28a-Nsp10 was constructed in a prokaryotic expression system. The high-efficiency expression of rNsp10 protein was achieved under induction with 1.5 mmol/L IPTG for 8 h at 18  to 37 ℃， with the protein mainly existing as inclusion bodies and purified yield of 15 mg/L. Immunization of C57BL/6J mice with rNsp10 elicited a strong humoral immune response， with a maximum antibody titer of 1∶409600， which remained at high levels during the later stages of immunization. Antibody isotype and immune response tests revealed that rNsp10 immunization not only induced a humoral immune response dominated by IgG1， but also significantly up-regulated the relative expressions of genes related to Th2 humoral immune response IL-4 and IL-10， as well as genes related to Th1 cellular immune response IL-6， IL-1β， IFN-γ， TNF-α， and IL-12 （P<0.01）. Reactogenicity test of the rNsp10 polyclonal antibody showed that mouse-derived anti-rNsp10 polyclonal antibodies could specifically recognize native Nsp10 protein expressed during PRRSV infection， demonstrating good reactogenicity.【Conclusion】 PRRSV Nsp10 is a hydrophilic protein containing 19 B-cell epitopes and is suitable for expression in a prokaryotic system. The rNsp10 protein exhibits strong immunogenicity， simultaneously capable of activating a humoral immune response dominated by IgG1 and a cellular immune response characterized by genes such as IFN-γ and IL-12. rNsp10 mouse-derived polyclonal antibodies specifically recognize native Nsp10 protein expressed during PRRSV infection.