The Expression And Activity Of Mutated Human CuZn-SOD In Synechococcus Sp.PCC7942

The Superoxide dismutase is an important oxygen free radical scavenger which catalyzes the superoxide anion radical disproportionation reaction. It was considered that O2- has serious damage to biological macromolecule and other cell components. SOD can prevent and treat the many kinds of diseases which causes by the superoxide radicals (such as familial amyotrophic lateral sclerosis, FALS). It has been used in some diseases such as inflammmation, tumor, skin disease,cardiovascular disease treatment and anti-senescence and so on. There are some patents to produce SOD cosmetics , anti-radiation SOD capsule, SOD preparation to treat the blood sickness . In some countries such as America, Japan and Western Europe, recombinant human SOD drugs were developed early, some of which have already reached stageâ…¡and stageâ…¢clinical study. It's considerated that SOD is a promising medicinal enzymes. As one of the health foods and the effective cosmeticd component, SOD has a widespread application prospect. However, the stability of SOD in vivo is relatively poor and the half-life is relatively short, so there are some limitations for SOD in fulfilling its functions.The human copper,zinc-superoxide dismutase (hCuZn-SOD) gene and its mutated one were separately cloned into a prokaryotic expression plasmid and expressed in E.coli. And then the activities of expressed hCuZn-SOD were determined at different temperatures and pH. Results show that the expressed recombinant of mutated hCuZn-SOD and normal hCuZn-SOD protein accounted for more than 45% of the bacteria's total protein and most of them existed as insoluble protein. The mutated hCuZn-SOD has higher activity than hCuZn-SOD. Furthermore the mutated hCuZn-SOD is stabler than hCuZn-SOD. From the perspective of gene mutation, the effort to improve the efficiency of enzyme is beneficient. The nucleotide sequence of cpc promoter was obtained by PCR. The expression vector pCMSOD which contained cpc promoter, hCuZn-SOD gene, rbc-polyA terminator and reporter gene (Km) was constructed. The pCMSOD plamid, in the end, was introduced into cells of Synechococcus sp.PCC7942 with homologous recombination platform, and transformants were screened by Kanamycin. Results of PCR and DNA sequence analysis showed that the target nucleotide had been genetically integrated into genome DNA of the host cell. After induced at 1200lx, the hCuZn-SOD protein was detected by SDS-PAGE and Western Blot in the transgenic Synechococcus sp. PCC7942. The result of protein scan showed that the aimed protein is 6.8% of the total dissoluble protein. The animal experiment results showed that the activities of Glutathione peroxidase (GSH-PX) in serum and the activities of Catalase (Cat) in blood were increased obviously; that the activity of SOD in serum and liver were increased markedly; that the content of Maleic Dialdehyde (MDA) in serum and liver were decreased obviously. It indicated that the transgenic Synechococcus sp.PCC7942 had obvious antioxidant effect in vivo.In this study the hCuZn-SOD gene and its mutated one were separately cloned into a prokaryotic expression plasmid and expressed in E.coli. And then the activity and stability of mutant hCuZn-SOD and hCuZn-SOD were compared. Then the mutant hCuZn-SOD was introduced into cells of Synechococcus sp.PCC7942 with homologous recombination platform. The animal experiment also showed that the transgenic Synechococcus sp.PCC7942 could enhance animal's antioxidative ability. The results of this study have certain theoretical significance and practical value. The study provide a basis for further research of oral hCuZn-SOD with much longger life.