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

生物技术通报 ›› 2012, Vol. 0 ›› Issue (12): 184-191.

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

王齐磊^{1, 2}, 李相前^{1, 2}, 徐琳寓^{1, 2}, 薛业敏³

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

收稿日期:2012-06-14 修回日期:2013-01-25 出版日期:2012-12-26 发布日期:2013-02-06
作者简介:王齐磊, 男, 硕士研究生, 研究方向:微生物分子生物学;E-mail:yshb318@163.com
基金资助:
：国家自然科学基金项目（20971050），江苏省科技支撑项目（BE2009651）

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

Wang Qilei^1,2, Li Xiangqian ^1,2, Xu Linyu^1,2, Xue Yemin³

1Faculty of Life Science & Chemical Engineering，Huaiyin Institute of Technology，Huai’an 223003 ；2Huaian High-Tech Research Institute of Nanjing University，Huai’an 223003 ；3Department of Food Science and Nutrition，Ginling College，Nanjing Normal University，Nanjing 210097

Received:2012-06-14 Revised:2013-01-25 Published:2012-12-26 Online:2013-02-06

摘要/Abstract

摘要： 超氧化物歧化酶（SOD）可以减轻超阳阴离子O₂^-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.coil 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。

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

Abstract: In this study, superoxide dismutaes genes fesod 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 OD₆₀₀ was higher than that of control. SDS-PAGE analysis showed that fesod 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-1 and their expression in E.coil.

王齐磊, 李相前, 徐琳寓, 薛业敏. 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[J]. 生物技术通报, 2012, 0(12): 184-191.

Wang Qilei, Li Xiangqian , Xu Linyu, Xue Yemin. AMB-1 Expression of Superoxide Dismutae Fe-SOD of Magnetospirillum magneticum AMB-1 in Escherichia coli and Its Enzymatic Characterization[J]. Biotechnology Bulletin, 2012, 0(12): 184-191.

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