| Summary: | 碩士 === 台北醫學院 === 醫學研究所 === 90 === Reactive oxygen species and other oxidants cause DNA damage, lipid peroxidation, protein denaturation, cell death and resultant organ injury. Substantial evidence suggests that oxygen free radicals are associated with not only aging process but also degenerative disorders, such as Parkinson’s disease, Amyotrophic Lateral Sclerosis, atherosclerosis and Rheumatoid Arthritis. Free radicals are also reported to be associated with malignancy, ischemia/reperfusion and inflammation. Manganese superoxide dismutase (Mn-SOD), an endogenous superoxide radical-scavenging mitochondrial enzyme, is crucial for protecting cells and tissues from oxidant injury and hyperoxia. Enhancement of endogenous cellular anti-oxidation capacity by introduction of Mn-SOD cDNA or full-length active Mn-SOD protein into cells may, therefore, offers attractive therapeutic strategy for diseases associated with oxidative stress. We therefore sought to sequence the human Mn-SOD gene and purify the active human Mn-SOD protein.
Human Mn-SOD gene library was established by reverse transcription and polymerase chain reaction (RT-PCR) of the total mRNA extracted from human liver tissue. Agarose gel electrophoresis indicated that Mn-SOD gene is a 667 base pairs DNA which encodes a 20 kDa protein. SOD expression vector was thus constructed and transformed into Escherichia coli (E. Coli) for gene expression. Western blotting analysis indicated that Mn-SOD could be over-expressed in the presence of IPTG. After isolation and purification, the enzyme activity of recombinant human Mn-SOD was assayed by using pyrogallo method. Our data indicated that Mn-SOD enzyme activity was Mn2+ ion concentration dependent. Baculovirus expression system was also employed to test the expression of Mn-SOD gene. Similar results were observed. We, therefore, concluded that sequencing of human Mn-SOD gene and synthesis of active recombinant human Mn-SOD protein are feasible and can contribute to future therapeutic strategies against diseases associated with oxidative stress.
|
|---|
