Prokaryotic Expression and Preparation of Polyclonal Antibody for Human SUMO-3 Gene

To construct a prokaryotic expression vector of Human SUMO-3,purify Human SUMO-3 proteins produced by the expression system,and prepare its antiserum.The fragment of human SUMO-3 gene was amplified from pEYPF-SUMO-3 plasmid by PCR.The enzyme digested target fragment was cloned into pET41a(+)clone and expression vector and transfected into E.coli.BL 21(DE3)plysS,in which SUMO-3 expression was induced by IPTG.After the soluble protein was purified through GST affinity chromatography,processed by identified by SDS-PAGE,a rabbit was immunized with the fusion protein,and the antiserum was obtained.The result of DNA sequence analysis showed that the cloned SUMO-3 gene sequence was completely corresponding to GenBank data.SDS-PAGE and Western blot showed that the expressed SUMO-3 fusion protein was about 44.0 kDa,mainly existing soluble protein of E.coli,that could be purified through GST affinity chromatography.The results of ELISA is positive,and Western blot confirmed that the antiserum reacted specifically to the SUMO-3 protein.A recombinant SUMO-3 protein and the specific polyclonal antibody have been obtained,which provides a basis for establishment of immunoassays of human SUMO-3.