Expression of mouse PD-1 extracellular region and its antibody preparation

Objective:To prepare mouse membrane PD-1 membrane extracellular region (mPD-1) and its antibody for studying the biological activity of PD-1.Methods:The recombinant protein mPD-1 was induced by IPTG in E.coli BL21(DE3) and the expressed protein was detected by SDS-PAGE and Western blot assay.The purified protein was used to immune rabbits to prepare polyclonal antibody,and the specificity and the titer of the antibody were detected with ELISA,Immunofluorescence assay and FCM.Results:The GST-mPD-1(Mr ≈42 000) protein could be expressed in E.coli BL21(DE3) with high efficiency.The recombinant protein was characterized with GST antibody by Western blot.Rabbit immunized with the purified protein produced high titer of antibody (titer ≈1:1 562 500).The PD-1 protein highly expressed in L929 cells was specifically combined with the antibody by cell immunofluorescence assay(CIF) and flow cytometry(FCM).Conclusion:Recombinant mPD-1 is expressed and purified with high antigenicity.The preparation of recombinant mPD-1 and its polyclonal antibody will lay the foundation for studying mPD-1 bioactivities.