趋磁菌AMB-1超氧化物歧化酶Fe-SOD在大肠杆菌中的表达及生理活性研究  

Expression of Superoxide Dismutae Fe-SOD of Magnetospinllum magneticum AMB-1 in Escherichia coli and Its Enzymatic Characterization

作　　者：王齐磊[1,2] 李相前[1,2] 徐琳寓[1,2] 薛业敏[3] 

机构地区：[1]淮阴工学院生命科学与化学工程学院,淮安223003 [2]南京大学淮安高新技术研究院,淮安223003 [3]南京师范大学金陵女子学院,南京210097

出　　处：《生物技术通报》2012年第12期184-191,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(20971050);江苏省科技支撑项目(BE2009651)

摘　　要：超氧化物歧化酶(SOD)可以减轻超阳阴离子O_2^-对细胞的毒害作用,提高菌体的抗氧化能力,从而改善菌株的生理状态。利用PCR技术,将趋磁菌AMB-1的超氧化物歧化酶基因fesod克隆至原核表达载体pET-20b(+),并在E.coli BL21(DE3)有效表达。重组菌株BL21(DE3)/(pET-20b-fesod-histag)在0.6 mmol/L IPTG诱导浓度下进行发酵培养,在生长前期2-14 h生长速率优于对照组E.coli BL21(DE3)/(pET-20b)。通过亲和层析柱纯化后,重组酶蛋白纯度达电泳均一,超氧化物歧化酶fesod活性最适作用温度为25℃,在25℃和45℃下酶热稳定较好,pH4.2-8.2之间酶活力稳定。趋磁菌AMB-1来源的Fe-SOD作为一种抗氧化酶,在大肠杆菌中的有效表达从一定程度上改善了宿主菌的生长情况。In this study, superoxide dismutaes genesfesod from Magnetospirillum AMB-1 were cloned and expressed in E.coil BL21 （ DE3 ） . The SOD gene was obtained from the genome of AMB-1 through PCR amplification, and the resulting amplified gene was cloned into an IPTG-inducible expression vector pET-20b, respectively. The expression vectors pET-20b-fesod-histag was further transformed into E.coil BL21 （ DE3 ） . In contrast to the control BL21 （ DE3 ） / （ pET-20b ） , the growth curves of the recominent strains showed that the growth rates of BL21 （ DE3 ） / （ pET-20b-fesod-histag ） was obviously higher than that of control. Superoxide dismutaes activity result showed that the SOD activity level of BL21 （DE3）/（ pET-20b-fesod-histag ） per OD600was higher than that of control. SDS-PAGE analysis showed thatfesod gene was expressed effectively and the expression product was about 22 kD. Moreover, the expression level of an hypothetical SOD protein derived from the host bacteria was gradually increasing during the IPTG inducible expression in BL21 （ DE3 ） / （ pET-20b ） . Molecular cloning of superoxide dismutaes genes from AMB-I and their expression in E.coli.

关 键 词：超氧化物歧化酶(SOD) 克隆表达 生理活性 酶学性质 趋磁菌AMB-1 

分 类 号：Q93[生物学—微生物学]