Preparation,Affinity Purification and Identification of Polyclonal Antibody Against Pig Annexin A2

The study was aimed at preparing and purifying polyclonal antibody of rabbit anti-pig ANNXA2 by GST-ANNXA2-coupled affinity chromatography.The fragment of pig Annexin A2 and EGFP fusion gene was amplified from pA2-EGFP plasmid by PCR.And then the fragment was subcloned into the prokaryotic expression vector pET-30a(+) to construct the recombinant plasmid p30a/AE,and expressed in inclusion body of E.coli.Rosetta(DE3) by IPTG induction.The fusion protein ANNXA2-EGFP which was purified by Ni-Resin HP affinity chromatography was used as immunogen to inject male rabbit to prepare polyclonal antibody anti-pig Annexin A2.The soluble fusion protein GST-ANNXA2 which has been prepared was coupled on NHS-activated Sepharose Fast Flow column,and then the polyclonal antibody was purified by this column.Purified antiserum titer amounted to 1:12 800 by indirect ELISA,and the purified antiserum was also demonstrated high specificity by Western blot and immunohistochemistry staining.The polyclonal antibody of rabbit anti-pig ANNXA2 was prepared and possessed high titer and high specificity,which laid the groundwork for the investigation of relationship between virus infection and pig Annexin A2.