Aim: To construct a prokaryotic expression vector of human SUMO-2, purify GST-SUMO2-SUMO2 fusion protein produced by the expression system, and prepare its antiserum.
Methods: The human SUMO-2 gene was amplified by PCR. The target fragment digested by the enzyme was cloned into a pET41a(+) expression vector and then transfected into E.coli. BL21 (DE3) pLysS, in which GST-SUMO2-SUMO2 fusion protein was induced by IPTG. After the soluble protein was purified by GST affinity chromatography and by identified by SDS-PAGE, the rabbits were immunized with the fusion protein and the antiserum was obtained.
Results: DNA sequence analysis showed the cloned SUMO-2 gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blot showed that the GST-SUMO2-SUMO2 fusion protein was about 52 kDa, which was mainly the soluble protein of E.coli and could be purified by GST affinity chromatography. The result of ELISA was positive and Western blot confirmed the antiserum reacted specifically to SUMO-2 protein.
Conclusion: SUMO-2 protein and its specific polyclonal antibody have been prepared, which provides a basis for the establishment of immunoassays of human SUMO-2.