翘嘴鳜铜锌超氧化物歧化酶重组蛋白表达、纯化及特性分析  

Analysis and characterization on recombinant protein of Cu/Zn superoxide dismutase from Siniperca chuatsi

作　　者：肖俊[1] 许亮清 胡向萍[1] 程周玉[1] 胡宝庆[1] 简少卿[1] 阳钢[1] 文春根[1] 

机构地区：[1]南昌大学生命科学学院,南昌330031 [2]南昌市科学院畜牧水产研究所,南昌330038

出　　处：《淡水渔业》2017年第2期11-17,共7页Freshwater Fisheries

基　　金：南昌市科技支撑计划项目(2012-KJ2C-NY-SC-002);国家自然基金(31472305;31460697;21467015);江西省教育厅项目(GJJ12024;GJJ10378);江西省自然科学基金(20132BAB204019)

摘　　要：超氧化物歧化酶(SOD)是清除生物体内超氧阴离子自由基的一种重要抗氧化酶。根据翘嘴鳜(Siniperca chuatsi)Cu/Zn-SOD基因序列(Gen Bank登录号:KJ558392.1)设计表达引物,扩增获得截去信号肽后的一段460 bp的序列,序列经过鉴定后,构建了重组表达质粒p ET-30a+Sc Cu/Zn-SOD,并将该质粒转入到BL21(DE3)中,用IPTG诱导进行表达。经过优化表达条件得到可溶的Sc Cu/Zn-SOD重组蛋白(rScCu/Zn-SOD),纯化重组蛋白后测定rScCu/Zn-SOD的浓度和酶活性。结果发现在20℃和37℃条件下均能够诱导Sc Cu/ZnSOD的表达。37℃时重组蛋白主要以包涵体形式存在。降低诱导温度和补充Cu^(2+)/Zn^(2+)可提高rScCu/Zn-SOD的表达量。在20℃、0.5 mmol/L IPTG条件下,添加0.5 mmol/L CuSO_4和0.1 mmol/L ZnCl_2于培养基中,重组蛋白的表达量明显升高。纯化后的重组蛋白浓度为0.14 mg/m L,酶活力为108.5 U/mg。rScCu/Zn-SOD最适温度为37℃,最适pH为7.0,可耐受5%浓度的SDS蛋白质变性剂。Superoxide dismutases （SODs） are one family of important antioxidant enzymes involved in scavenging superox-ide anion free radical in organisms. In the present paper, the expression primer was designed by Cu/Zn - SOD gene se-quence of Siniperca chuatsi （named as ScCu/Zn - SOD, accession numbers： KJ558392. 1 ） . A truncated signal peptide sequence with 460 bp was amplified. After identification of the sequence, the constructed recombinant expression plasmid （pET - 30a ＋ ScCu/Zn - SOD） was transformed into BL21（DE3） , and was expressed by induction with IPTG. The solu-ble recombinant protein of ScCu/Zn - SOD （ designated as rScCu/Zn - SOD） was obtained by optimized expression condi-tion, and the concentration and activity of rScCu/Zn - SOD was measured after the purification of recombinant protein. The results showed that the expression of ScCu/Zn - SOD could be induced under 20 ℃and 37℃ conditions. The rScCu/Zn - SOD was mainly aggregated to form inclusion bodies at 37℃ The expression quantity of rScCu/Zn - SOD could be im-proved by reduction induction temperature and supplement Cu2 ＋ /Zn2 ＋ . The addition of 0. 5 mmol/L CuS04 and 0. 1 mmol/ L ZnCl2 in culture medium was optimal under 20 and 0. 5 mmol/L IPTG condition, and the expression quantity of rSc-Cu/Zn - SOD was obviously increased. The results can provide basic data for the research on functional characterization ofSOD protein.

关 键 词：翘嘴鳜(Siniperca chuatsi) 铜锌超氧化物歧化酶 原核表达 蛋白特性 

分 类 号：S917.4[农业科学—水产科学]