The Function And Characteration Of Cu,Zn Superoxide Dismutase Of The Sea Cucumber(Apostichopus Japonicus)

Copper zinc superoxide dismutase(SOD1)is an important enzyme for defense against oxidative damage in organism,which has an important antioxidant protective effect on organism.In this study,the function and characteration of Cu,Zn superoxide dismutase of the sea cucumber were studied.The Aj-SOD1 protein was predicted by bioinformatics analysis(pI=5.65,MW=15.47 kDa).Phylogenetic tree of Aj-SOD1 was constructed by homology comparison.The analysis of the structure showed that the target protein was able to bind the zinc and copper ions.It has demonstrated that Aj-SOD1 was expressed in all tissues,but the expression was higher in intestinal and respiratory tree.In vitro,it showed that the Aj-SOD1 protein was up-regulated in the course of H2O2/LPS stimulation.At the same time,the repair function of SOD1 protein from sea cucumber on H2O2/LPS cells was studied.Then we cloned the full length coding sequence of copper and zinc superoxide dismutase(Aj-SOD1).The coding sequence of Aj-SOD1 gene was obtained by the cDNA template coming from the reverse transcription PCR amplification.The cloning vector pMD18-Aj-SOD1 with the target gene at both ends(Xho I/Nde I restriction sites,and His tag)was constructed.Then the clone plasmid and expression plasmid pET30a(+)were digested respectively to construct the recombinant expression vector pET30a-Aj-SOD1.The vector pET30a-Aj-SOD1 was transformed into E.coli BL21(DE3)competent cells.The SOD1 protein was expressed by the IPTG,it showed that the protein was high purity,solubility.The optimal conditions for inducing the expression of the protein were optimized.The optimal expression temperature was 37 ?,the induction time was 8 h,the speed was set at 200 r/min,concentration of inducer was 0.1 mM.Then we got the high purity protein by the Ni-NTA.This study plays an important role in studying the properties of copper,zinc superoxide dismutase,It also plays an important guiding role in the development and breeding of sea cucumber.