Cloning and expression of the Mn-SOD gene from Phascolosoma esculenta

Manganese superoxide dismutase (Mn-SOD; SOD(2)) is an important antioxidant defense enzyme. In this study, a full-length Mn-SOD cDNA was cloned from the cDNA library of Phascolosoma esculenta. The cDNA is 1385 bp in length, including an open reading frame (ORF) of 681 bp encoding 226 amino acids. The predicted protein has a calculated molecular weight of 25.2 kDa and a theoretical isoelectric point of 5.96. BLAST analysis revealed the predicted protein shared 70% identity with homologues in Caenorhabditis elegans and Gallus gallus. The SOD gene was inserted into Escherichia coli expression plasmid pET-28a (+) to produce pET-SOD. The recombinant Mn-SOD of P. esculenta was expressed following IPTG induction, and verified by Western blot analysis using antiserum from immunized mice. Furthermore, fluorescent real-time PCR analysis revealed varying degrees of induction of SOD(2) mRNA expression in the blood of P. esculenta exposed to heavy metals (Cd(2+), Cu(2+) and Zn(2+)) and thermal stresses (4 degrees C and 37 degrees C).