嗜热嗜热菌超氧化物歧化酶的克隆、表达与鉴定

Cloning,expression and identification of superoxide dismutase from Thermus thermophilus wl

从厦门近海温泉中分离并鉴定了一株嗜热嗜热菌(Thermus thermophiluswl).将从基因组中克隆到的超氧化物歧化酶(supseroxide dismutase,SOD)基因插入pGEX-4T-2表达载体,在E.coliDH5α中成功表达了谷胱甘肽-S-转移酶(glutathione S-trans-ferase,GST)融合的目的蛋白.重组SOD酶的比活性达580.3U/mg.体外表达纯化的GST-SOD蛋白免疫小鼠,制备SOD特异性抗体.Western blot结果显示,鼠抗GST-SOD抗体能特异性地与T.thermophiluswl的细胞裂解液中一分子量为27 kDa的蛋白反应,与该基因ORF预期大小接近.以上研究为进一步研究该酶的生理生化特性奠定了基础.

A Thermus thermophilus wl strain was isolated from hot spring in the Xiamen offing. The supsroxide dismutase （SOD） gene cloned from the genome was cloned into pGEX-4T-2 vector and expressed as fusion protein with glutathione S-transferase （GST） in Escherichia coli strain DH5α. The specific activity of fusion protein amounted to 580.3U/mg. Specific antibody was raised using fusion protein GST-SOD. Western blot analysis showed that the mouse anti-GST-SOD antibody reacted specifically with a 27kDa protein present in the cell extract of T. thermophilus wl,which is close to the ORF of SOD. These data laid a foundation for the further study on the biochemical and physiological characteristics of this enzyme.