Efficiency Expression Of Manganese Superoxide Dismutase Gene From Soybean By Food-grade Vector In Lactococcus Lactis

Superoxide dismutase (SOD) is an important defensive enzymes for superoxide anionradicals of organism. They have a widely use in resisting inflammation, senility, radiation andvirus in organism. SODs are widely used in food, chemical and pharmaceutical industrialproduction. Manganese superoxide dismutase (MnSOD) is one of the SOD. A plenty ofresearch shows that, over-expression of MnSOD gene in organism has associated with a varietyof tumors. It has been recognized as a new type antioncogene and attracted widespreadattention. However, expression of MnSOD from organisms is lower in vivo. It is too difficult toobtain a large number of MnSOD by traditional fermentation. Application of geneticengineering techniques, to obtain an high-efficient expression of recombinant strains forfermenting of MnSOD, has become a research hotspot.A pair of gene-specific primer is design and synthesis based on the published sequences ofMnSOD from soybean. Open reading frame of MnSOD was cloned to the PMD18-T vectorfrom the recombinant cloning vector pREP5N-MnSOD. Sequence analysis of target fragmentshows it corresponds with the soybean gene sequence published in GenBank and100%homology. Target fragment has a726bp length. Then target fragments were cloned into thevector pNZ8149and reformed plasmid pNZS to construct the food-grade vector. Therecombinant plasmids then transformed into L. lactis NZ3900strain by electronic perforation,and the growth ability of the transformants was detected on Elliker medium. The recombinantplasmids named pNZ-SOD and pNZS-SOD were thus constructed successfully after PCRamplification, lingation and identification.Induced L. lactis NZ3900/pNZ-SOD and L. lactisNZ3900/pNZS-SOD’s expression by nisin and tnen analysis the expressed products of them bySDS-PAGE. The SOD enzymatic activity of the transformant L. lactis NZ3900/pNZ-SOD andL. lactis NZ3900/pNZ-SOD were determined by the inhibition of nitrotetrazolium. The SODenzymatic activities of the transformant L. lactis NZ3900/pNZS-SOD was1.6times higherthan that of the transformant L. lactis NZ3900/pNZS-SOD，13.5times higher than that of theparent strain. MnSOD was enhancd expression in L. lactis NZ3900/pNZS. Subsequently apreliminary analysis for enzymatic propertiesof MnSOD which L. lactis NZ3900/pNZS-SODexpression strain secreted into the extracellular. Secreted expression product’s thermal stability(has the best activity at90℃), half-life (90℃,28min), acid and alkali resistance, tolerance topronase E are similar with known MnSOD. It provide a theoretical basis for efficiently express MnSOD in Lactococcus lactis bacteria and direct application of expresstion in the food anddrug industry.