Radioprotective Effects Of MnSOD Transduced By A Cell-penetrating Peptide

Manganese superoxide dismutase(MnSOD)can reduce the oxidative damage caused by free radicals.It is synthesized as an inactive zymogen and converts to the mature form after the cell permeable leading peptide is cleaved.But mature MnSOD can't enter into cells.The DNA fragment containing the full nucleotide of full-length human MTS-MnSOD was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with GST tag.DNA construct was then transformed into Escherichia coli BL21(DE3)and expression was induced with IPTG at 25 ? for 5 h.GST-MTS-MnSOD(46 kDa)was purified subsequently with ultrasonication of bacterial lysate and GST resin column affinity chromatograpHy.SOD activities of GST-MTS-MnSOD were 1788U/mg.Then,GST tag was cleaved with thrombin,and a crude MTS-MnSOD recombinant protein(25 kDa)was obtained and further purified by heparin affinity chromatograpHy.SOD activity of MTS-MnSOD was 2 000 U/mg.The result of the research on the two proteins' temperature and pH stability showed that both temperature and pH have major effects on the proteins,however,they both maintained relatively high enzymatic activity under the biological condition(37 ?,pH 7.0),and MTS-MnSOD showed higher stability than GST-MTS-MnSOD.In addition,both MTS-MnSOD and GST-MTS-MnSOD were proved to be cell permeable and nontoxic by the method of FITC labeling and WST respectively.Furthermore,the protective effect of two proteins on 6 Gy whole body irradiated mice was investigated.In this study,a significant deficit in the body weight,the number of peripheral white blood cells,hepatic antioxidant system,and shrunken spleen and thymus were observed in mice of the X-ray irradiation alone group on the 8th day after radiotherapy.Amifostine pretreatment significantly(P<0.05)raised peripHeral blood white cell counts of the mice,reduced hepatic MDA level,and increased hepatic GSH-Px,SOD and T-AOC activities.GST-MTS-MnSOD fusion protein had a prominent protective effect on the immune system.It could significantly improve the spleen index and thymus index of irradiated mice(P<0.05).MTS-MnSOD pretreatment was superior to GST-MTS-MnSOD pretreatment in many ways.Pretreated of 0.4 ?M MTS-MnSOD showed equivalent protection effect as Amifostine.Pretreatment with 0.2 ?M MTS-MnSOD showed the best protective effect as it significantly(P<0.05)raised the body weight and the hepatic antioxidant ability of irradiated mice.It did be protective for other irradiated damage too although there was no significance.