杨梅铜锌超氧化物歧化酶基因(MrCu/Zn-SOD1)的原核表达及活性鉴定

Prokaryotic Expression of Copper Zinc-Superoxide Dismutase Gene(MrCu/Zn-SOD1) from Morella rubra and its Enzymatic Activity Detection

为获得高表达杨梅(Morella rubra)铜锌超氧化物歧化酶(MrCu/Zn-SOD1)的工程菌,本实验采用RT-PCR技术从杨梅果实中分离扩增了MrCu/Zn-SOD1的cDNA序列(456bp),将该基因重组到原核表达载体pGEX-2T中,酶切、测序分析表明,重组质粒pGEX-MrCu/Zn-SOD1结构正确。重组质粒转化大肠杆菌BL21(DE3)进行诱导表达。IPTG诱导表达分子量约41kD融合蛋白GST-MrCu/Zn-SOD1。诱导表达后的菌体超声裂解液经谷胱甘肽亲和层析纯化,得到高纯度的GST-MrCu/Zn-SOD1。采用氯化硝基四氮唑蓝法和黄嘌呤氧化法分析其活性。结果表明,GST-MrCu/Zn-SOD1具有特异性SOD酶活性。

To obtain high expression plasmid of Morella rubra with Cu, Zn superoxide dismutase 1 （MrCu/Zn- SOD1）, RT-PCR was used in this experiment to isolate and amplify the cDNA sequence of MrCu/Zn-SODI （456 bp） from Morella rubra and was inserted into pGEX-2T vector. Enzyme digestion and sequence analysis showed that recombinant plasmid of pGEX-MrCu/Zn-SOD1 structure was correct. The E. coli BL21 （DE3） cells were transformed by the recombinant plasmid. The molecular weight of GST-MrCu/Zn-SOD1 fusion protein induced by IPTG was around 41 kD. High purity of GST-MrCu/Zn-SOD 1 was obtained after ultrasonic decomposi- tion of liquid and the affinity purification through Glutathione Sepharose 4B column. The enzymatic activity of this product was determined by the nitroblue tetrazolium assay and Xanthine oxidation method. The results showed that enzymatic activity of GST-MrCu/Zn-SOD 1 had specificity.