大鼠晶体上皮细胞Cu-ZnSOD的分子克隆表达和活性鉴定

Cloning,expression and biological activity identification of Cu-Zu SOD from rat epithelial cells of lens

不同来源的超氧化物歧化酶（SOD）的克隆已有报道，用RT－PCR方法我们成功地从大鼠晶体上皮细胞中克隆出了CU－ZnSODcDNA并利用pMal载体在大肠杆菌中高效表达出MBP－SOD融合蛋白可占菌体总蛋白40％左右，采用的蛋白纯化系统的亲和层析柱就是利用的MBP融合蛋白可被快速纯化而设计的，实验证明此方法可快捷、高效地表达有生物学活性的MBP—SOD融合蛋白。

Superoxide dismutase scavenges toxic superoxide anion produced during normal metabolism or after oxidative insult. Altered levels of SOD have been implicated in multistage cancinogenesis and other pathological status. Here we report that a full length cDNA of rat Cu-Zn SOD has been successfully cloned by RT-PCR and Nucletide sequence bas been confirmed by Sanger's method. Subsequently, the SOD gene was recombined into an expression of vector pMal,inserted upstream MBP gene and expressed in E. coli as a fusion protein (MBP-SOD protein) induced by IPTG. The recombinant rat SOD was purified to homogeneity by this single step purification. We identified the activity of SOD protein by examining pyrogallol self-Oxidation velocity. This investigation certainly would facilitate the study of SOD and its potential therapeutical uses.