中国生物工程杂志

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 blotting 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 blotting 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.