Polyclonal Antibody Preparation And Identification Of Human SUMO-1 Protein And The Subcellular Localization Of SUMO-1

Objective: As a protein similar to the modified protein ubiquitin,SUMO (small ubiquitin-related modifier) gradually becomes a international research hotspot. In order to further study the function of SUMO-1 protein, we constructed SUMO-1 gene eukaryotic and prokaryotic expression systems, prepared polyclonal antibody for human SUMO-1 protein, observed its sub-cellular localization, and made a preliminary research on the interaction between SUMO-1 and PML.Methods: The coding C-terminal 97 amino acids fragment of human SUMO-1 gene was amplified from pcDNA-HA-GG-SUMO1 plasmid by PCR and constructed prokaryotic expression vector pET41a (+)-SUMO1 (pET-S1) , pET41a (+)-SUMO1-SUMO1 (pET-SS1) and eukaryotic expression vector pCMV-HA-SUMO1 (HA-S1) , pCMV-Myc-SUMO1 (Myc-S1) , pEGFP-N1-SUMO1 (GFP-S1) , pDsRed-N1-SUMO1 (Red-S1) respectively. The eukaryotic expression vectors were transformed into E. coli BL21 (DE3) pLysS, and then the expression of fusion protein GST-SUMO1 (GST-S1) and GST-SUMO1-SUMO1 (GST-SS1) was induced by IPTG. The fusion protein GST-S1 and GST-SS1 was purified through GST affinity chromatography, rabbits were immunized with the purified protein, and the antiserum was obtained, processed by ELISA, Western-blotting and immunofluorescence to detect the sensitivity and specificity of its antibody. Eukaryotic expression vectors GFP-S1 and Red-S1 were respectively transfected into Hela cell by liposome and expressed transiently, then we observed sub-cellular localization of protein SUMO-1 by fluorescence microscope. Eukaryotic expression vectors Red-S1 and pEGFP-PML were transfected into Hela cell by liposome and expressed transiently, then we observed the co-localization of them by fluorescence microscope.Results: (1) SUMO-1 prokaryotic expression vectors pET-S1, pET-SS1 and eukaryotic expression vectors HA-S1, Myc-S1, GFP-S1and Red-S1were constructed successfully.(2) Fusion protein GST-SS1 and GST-S1 which induced by IPTG expressed successfully, and purified fusion protein GST-SS1, GST-S1 and human SUMO-1 protein.(3) Polyclonal antibody for human SUMO-1 was obtained by rabbits which were immunized with the fusion protein GST-S1 and GST-SS1 as antigen respectively. By comparing the polyclonal antibody titer, we found that the series of antigen can effectively improve their polyclonal antibody titer. The polyclonal antibody can be used for test the endogenetic SUMO-1 protein in Hela.(4) The vectors Myc-S1/ GFP-S1/Red-S1were expressed in Hela successfully and we found that Human SUMO-1 protein was localized in the nucleus.(5) Eukaryotic expression vectors Red-S1 and pEGFP-PML were transfected into Hela cell by liposome and expressed transiently. PML and SUMO-1 co-localize in the nucleus was observed.Conclusions: (1) Human SUMO-1 gene prokaryotic and eukaryotic expression system were constructed successfully.(2) Specific and high- titer polyclonal antibody for SUMO-1 was prepared successfully.(3) The series of antigen can effectively improve their polyclonal antibody titer.(4) SUMO-1and PML co-localize in the nucleus indicate to PML is a substrate of SUMO-1 protein again.