Molecular Cloning And MRNA Expression And Recombinant Protein Of An Intracellular Cu/Zn-superoxide Dismutase(Cu/Zn-SOD) Gene From Hard Clam (Meretrix Meretrix)

Cu/Zn superoxide dismutase (Cu/Zn-SOD) involves in many biological events in the organisms, not only in scavenging oxygen free radicals, molecular damage and body aging defense, but also involved in signal transduction and neural survival. Cu/Zn-SOD is the most important enzyme in the active oxygen scavenging enzymes. In the present study, the intracellular Cu/Zn-SOD gene of hard clam (Meretrix meretrix) was cloned through SMART cDNA libraries and RACE approaches. The full-length cDNA of Mm-Cu/Zn-SOD was 1383 base pares (bp) with a 462 bp open reading frame encoding 153 amino acids. The deduced amino acid of hard clam Cu/Zn-SOD shared high similarity with the Cu/Zn-SODs from other species, indicating that Cu/Zn-SOD should be a member of intracellular Cu/Zn-SOD family. Several highly conserved motifs including Cu/Zn binding sites (H(46), H(48), H(63), H(119) for Cu binding, and H(63), H(71), H(80), D(83) for Zn binding), intracellular disulfide bond and two Cu, Zn SOD signatures were also identified in Cu/ZnSOD. The main spatial structure of protein isβ- barrel by tertiary structure prediction. The Cu/Zn-SOD this hard clam is highly homologous to Cu/Zn-SOD in Crassostrea ariakensis and other nine shellfish. The results of real-time PCR showed that the lowest expression of Cu/Zn-SOD was detected in the hard clam without Vibrio anguillarum challenged, and the expression increased as time increased, the highest expression level was found in the 6 hours after challenged with Vibrio anguillarum, then began to decrease, until the 32 hours to stabilize, which showed that gene Cu/Zn-SOD participated in the elimination of superoxide anion radical during the challenge with the bacteria. The recombinant protein of Cu/Zn-SOD gene in hard clam was also successfully expressed in E. coli. The expressed recombinant protein showed biological activity, maintained 60 percent of biological activity under 10-30℃and completely inactivated until 90℃. This recombinant protein showed highest activity under pH7.4, and deactivated at pH12.2, so the Cu/Zn-SOD is a stable enzyme.