Polyclonal Antibody Preparation Of Human SUMO2 Gene And The Study Of Its Subcellular Localization

SUMO is a highly conserved, small ubiquitin-related modifier that has been shown to be covalently conjugated to variety of cellular proteins. There are four members of the SUMO family described in vertebrates: SUMO1,SUMO2,SUMO3 and SUMO4. It has remarkably similar secondary structure with ubiquitin and is covalently conjugated to other proteins in a similar multistep process to ubiquitination. However unlike the ubiquitin system, which primarily targets substrate proteins to the proteasome for degradation, sumoylation participates in a number of cellular processes such as nuclear transport, transcriptional regulation, apoptosis and cell cycle control. Recent research revealed that sumoylation is involved in mitochondrial fission, DNA repair and the ion channel control. Many diseases or disorders are shown to be related to sumoylation/de sumoylation cycle.In the SUMO family, SUMO1 belongs to the primary family which has already been investigated very much while SUMO2 belongs to the secondary family with only a little study. In the experiment, the primer which specifies homo SUMO2 gene was designed according to its sequence reported previously. SUMO2 fragment was obtained by PCR with the recombinant plasmid pEYFP-SUMO2 as the template. The PCR products were cloned into pET41a vector and the correct recombinant plasmids pET41a-SUMO2-SUMO2 was gained. The prokaryotic expression construct for SUMO2 was transformed into BL21(DE3) plysS, and then recombinant engineering bacteria were selected and identified. Culture engineering bacteria in the gross and add 1mM IPTG to induce foreign protein expression. The cells were harvested by centrifugation. Then collected the soluble protein and purified the protein with GST affinity chromatography. Purified recombinant protein was analyzed by SDS-PAGE. In order to get antibody, the rabbits were immunized with the fusion protein GST-SUMO2-SUMO2 according to the routine method and then collected the serum. The titer of the rabbit anti-SUMO2 antibody was measured by ELISA assay and the specificity of rabbit anti-SUMO2 antibody was measured by Western blotting. In order to further validate our rabbit anti-SUMO2 antibody, we have also used it to detect the endogenesis SUMO2 in Hela through the Western blotting. In addition, we also construct the SUMO2 eukaryotic vectors including pCMV-HA-SUMO2(HA-S2),pCMV-Myc-SUMO2(Myc-S2),pEGFP-SUMO2(G-S2) and pDsRed-SUMO2(R-S2), which could be used to observe the subcellular localization of SUMO2 and the localization between SUMO2 and other regulation factors.The results showed that the SUMO2 gene was successfully subcloned from pEYFP-SUMO2. BL21(DE3) plysS strains with pET41a-SUMO2-SUMO2 constructs successfully expressed the recombinant protein bearing an GST tag with molecular weight of 52 kDa which is consistent with the predicted putative molecular weights of the fusion protein GST-SUMO2-SUMO2. T'he recombinant protein was proved to be the specific homo SUMO2 protein with western-blotting using the Rabbit anti-SUMO2 IgG HPR conjugated as the antibody. The anti-serum was proved to be the specific anti-SUMO2 with the Western-blotting using the anti-serum as the first antibody. ELISA showed that the titer of the rabbit anti-SUMO2 antibody is above 1: 40 000. Meanwhile, we also detect the endogenesis SUMO2 in Hela and elementary observe the subcellular localization of SUMO2.In conclusion, the experiment laid a firm foundation for the further research of the interact and colocalization between SUMO2 and other proteins.