Preparation and application of polyclonal antibody against porcine GSDMD protein
In order to prepare porcine GSDMD polyclonal antibody,the recombinant expression plasmid pET-21a-nGSDMD was constructed,and the recombinant nGSDMD protein was successfully expressed and purified.nGSDMD polyclonal antibody(pAb)was produced by immunizing New Zealand white rabbits with purified recombinant protein.The antibody titre was 1∶64 000 by ELISA.Western-blot and IFA showed that the antibody could specifically recognize Flag-GSDMD protein expressed in HEK293T cells and GSDMD protein expressed in alveolar macrophages(PAMs)after PRRSV infection.The polyclonal antibody has good specificity and no cross-reactivity.It can identify and detect GSDMD fragments under the condition of viral infection and can be used to detect PRV and PRRSV-induced pyroptosis.In conclusion,this study provides a good tool for exploring GSDMD protein and lays a foundation for further exploring the biological function of GSDMD protein.
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