Production of functional human CuZn-SOD and EC-SOD in bitransgenic cloned goat milk.

Abstract

Human copper/zinc superoxide dismutase (CuZn-SOD) and extracellular superoxide dismutase (EC-SOD) are two superoxide dismutases that scavenge reactive oxygen species (ROS). Their biological role of eliminating oxidative stress caused by excessive ROS levels in living organisms has been utilized in medical treatment, preventing skin photoaging and food preservation. In this study, we employed two sequences that encode human CuZn-SOD and EC-SOD, along with goat beta-casein 5' and 3' regulatory elements, to construct mammary gland-specific expression vectors. Bitransgenic goats were generated using somatic cell nuclear transfer (SCNT), which employed co-transfection to generate bitransgenic goat fetal fibroblast cells as donor cells, and the expression of human CuZn-SOD and EC-SOD and their biological activities were assayed in the milk. PCR and Southern blot analysis confirmed that the cloned goat harbors both hCuZn-SOD and hEC-SOD transgenes. rhCuZn-SOD and rhEC-SOD were expressed in the mammary glands of bitransgenic goat, as determined by western blotting. The expression levels were 100.14 ± 5.09mg/L for rhCuZn-SOD and 279.10 ± 5.38mg/L for rhEC-SOD, as determined using ELISA. A total superoxide dismutase assay with WST-8 indicates that the biological activity of rhCuZn-SOD and rhEC-SOD in goat milk is 1451 ± 136 U/mL. The results indicate that two expression vectors can simultaneously transfect goat fetal fibroblast cells as donor cells to produce transgenic goats by SCNT, and the CuZn-SOD and EC-SOD proteins secreted in the mammary glands showed biological activity. The present study thus describes an initial step in the production of recombinant human SODs that may potentially be used for therapeutic purposes.