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
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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 superoxide anion free radical in organisms. In the present paper,the expression primer was designed by Cu/Zn-SOD gene sequence 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( p ET-30 a + Sc Cu/Zn-SOD) was transformed into BL21( DE3),and was expressed by induction with IPTG. The soluble recombinant protein of ScCu/Zn-SOD( designated as rScCu/Zn-SOD) was obtained by optimized expression condition,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 improved by reduction induction temperature and supplement Cu~(2+)/Zn~(2+). The addition of 0. 5 mmol/L CuSO_4 and 0. 1 mmol/L ZnCl_2 in culture medium was optimal under 20 ℃ and 0. 5 mmol/L IPTG condition,and the expression quantity of r ScCu/Zn-SOD was obviously increased. The results can provide basic data for the research on functional characterization of SOD protein.
Superoxide dismutases( SODs) are one family of important antioxidant enzymes involved in scavenging superoxide anion free radical in organisms. In the present paper,the expression primer was designed by Cu/Zn-SOD gene sequence 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( p ET-30 a + Sc Cu/Zn-SOD) was transformed into BL21( DE3),and was expressed by induction with IPTG. The soluble recombinant protein of ScCu/Zn-SOD( designated as rScCu/Zn-SOD) was obtained by optimized expression condition,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 improved by reduction induction temperature and supplement Cu~(2+)/Zn~(2+). The addition of 0. 5 mmol/L CuSO_4 and 0. 1 mmol/L ZnCl_2 in culture medium was optimal under 20 ℃ and 0. 5 mmol/L IPTG condition,and the expression quantity of r ScCu/Zn-SOD was obviously increased. The results can provide basic data for the research on functional characterization of SOD protein.
引文
[1]迟春萍,陈子杨,徐军,等.毕赤酵母表达铜锌超氧化物歧化酶中试工艺研究[J].中国兽医学报,2012,32(2):267-271.
[2]Fridovich I.Superoxide dismutases[J].Annu Rev Biochem,1975,44:147-159.
[3]Fridovich I.Superoxide radical and superoxide dismutases[J].Annu Rev Biochem,1995,64(1):97-112.
[4]Mruk D D,Silvestrini B,Mo M Y,et al.Antioxidant superoxide dismutase-a review:its function,regulation in the testis,and role in male fertility[J].Contraception,2002,65(4):305-311.
[5]Whitfield A K,Elliott M.Fishes as indicators of environmental and ecological changes within estuaries:a review of progress and some suggestions for the future[J].J Fish Biol,2002,61(Supplement s A):229-250.
[6]Marikovsky M,Ziv V,Nevo N,et al.Cu/Zn superoxide dismutase plays important role in immune response[J].J Immunol,2003,170(6):2993-3001.
[7]Ken C F,Lin C T,Shaw J F,et al.Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress[J].Mar Biotechnol,2003,5(2):167-173.
[8]Li Z,Liang H W,Li L,et al.Molecular cloning and differential expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Hypophthalmichthys molitrix[J].Fish Shellfish Immunol,2011,30(2):473-479.
[9]Kim K Y,Sang Y L,Cho Y S,et al.Molecular characterization and mRNA expression during metal exposure and thermal stress of copper/zinc-and manganese-superoxide dismutases in disk abalone,Haliotis discus discus[J].Fish Shellfish Immunol,2007,23(5):1043-1059.
[10]Yang J,Dong S,Jiang Q,et al.Characterization and expression of cytoplasmic copper/zinc superoxide dismutase(Cu Zn SOD)gene under temperature and hydrogen peroxide(H2O2)in rotifer Brachionus calyciflorus[J].Gene,2013,518(2):388-396.
[11]Dalziel J E,Shyan W S,Thai P,et al.Expression of human BK ion channels in Sf9 cells,their purification using metal affinity chromatography,and functional reconstitution into planar lipid bilayers[J].J Chromatogr B,2007,857(2):315-321.
[12]Santovito G,Cassini A,Piccinni E.Cu,Zn superoxide dismutase from Trematomus bernacchii:Functional conservation and erratic molecular evolution in Antarctic teleosts[J].Comp Biochem Physiol Part C Toxicol Pharmacol,2006,143(143):444-454.
[13]Ken C F,Lin C T,Shaw J F,et al.Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress[J].Mar Biotechnol,2003,5(2):167-173.
[14]Lin C T,Lee T L,Duan K J,et al.Molecular cloning,characterization,and expression of a c DNA coding Copper/Zinc superoxide dismutase from black porgy[J].J Agric Food Chem,2000,48(9):4444-4447.
[15]Li Z,Liang H W,Li L,et al.Molecular cloning and differential expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Hypophthalmichthys molitrix[J].Fish Shellfish Immunol,2011,30(2):473-479.
[16]Tian J,Sun B,Luo Y,et al.Distribution of Ig M,Ig D and Ig Z in mandarin fish,Siniperca chuatsi,lymphoid tissues and their transcriptional changes after Flavobacterium columnare,stimulation[J].Aquaculture,2009,288(1):14-21.
[17]Wang G,Li J,Zou P,et al.Expression pattern,promoter activity and bactericidal property ofβ-defensin from the mandarin fish Siniperca chuatsi[J].Fish Shellfish Immunol,2012,33(3):522-531.
[18]Sun B J,Wang G L,Xie H X,et al.Gene structure of goosetype lysozyme in the mandarin fish Siniperca chuatsi,with analysis on the lytic activity of its recombinant in Escherichia coli[J].Aquaculture,2006,252(2–4):106-113.
[19]Xiao J,Zhou Z C,Chen C,et al.Tumor necrosis factor-alpha gene from mandarin fish,Siniperca chuatsi:Molecular cloning,cytotoxicity analysis and expression profile[J].Mol Immunol,2007,44(14):3615-3622.
[20]程周玉,文春根,胡向萍,等.翘嘴鳜铜锌超氧化物歧化酶基因的分子克隆与原核表达[J].南昌大学学报(理科版),2015,39(6):587-596.
[21]Xu H H,Ma H,Hu B Q,et al.Molecular cloning,identification and functional characterization of a novel intracellular Cu-Zn superoxide dismutase from the freshwater mussel Cristaria plicata[J].Fish Shellfish Immunol,2010,29(4):615-622.
[22]Bao Y,Li L,Xu F,et al.Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians:Its gene structure,mRNA expression and recombinant protein[J].Fish Shellfish Immunol,2009,27(2):210-220.
[23]刘建荣,赵晓瑜,宋小青,等.重组人Cu,Zn-SOD包含体的复性、纯化及复性蛋白稳定性研究[J].中国药学杂志,2007,42(13):969-974.
[24]龙建银,王会信.外源基因在大肠杆菌中表达的研究进展[J].生物化学与生物物理进展,1997(2):126-132.
[25]Dalziel J E,Shyan W S,Thai P,et al.Expression of human BK ion channels in Sf9 cells,their purification using metal affinity chromatography,and functional reconstitution into planar lipid bilayers[J].J Chromatogr B,2007,857(2):315-321.
[26]田春美,钟秋平.超氧化物歧化酶的现状研究进展[J].中国热带医学,2005,5(8):1730-1732.
[27]吴燕燕,李来好,郝志明,等.罗非鱼肝脏中超氧化物歧化酶的提取、纯化与分析[J].水产学报,2007,31(4):518-524.
[28]张尔贤,李建喜.鲨鱼肝超氧化物歧化酶的纯化与部分性质研究[J].中国药学杂志,1999(4):231-233.
[29]梅光泉,廊惠芳.超氧化物歧化酶中微量元素的化学行为和生物学功效[J].微量元素与健康研究,2003,20(5):59-62.
[30]Salin M L,Oesterhelt D.Purification of a manganese-containing superoxide dismutase from Halobacterium halobium[J].Arch Biochem Biophys,1988,260(2):806-810.
[31]王伟伟,刘存岐,李道季.3种虾超氧化物歧化酶部分理化性质比较[J].水产科学,2009,28(4):200-204.
[2]Fridovich I.Superoxide dismutases[J].Annu Rev Biochem,1975,44:147-159.
[3]Fridovich I.Superoxide radical and superoxide dismutases[J].Annu Rev Biochem,1995,64(1):97-112.
[4]Mruk D D,Silvestrini B,Mo M Y,et al.Antioxidant superoxide dismutase-a review:its function,regulation in the testis,and role in male fertility[J].Contraception,2002,65(4):305-311.
[5]Whitfield A K,Elliott M.Fishes as indicators of environmental and ecological changes within estuaries:a review of progress and some suggestions for the future[J].J Fish Biol,2002,61(Supplement s A):229-250.
[6]Marikovsky M,Ziv V,Nevo N,et al.Cu/Zn superoxide dismutase plays important role in immune response[J].J Immunol,2003,170(6):2993-3001.
[7]Ken C F,Lin C T,Shaw J F,et al.Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress[J].Mar Biotechnol,2003,5(2):167-173.
[8]Li Z,Liang H W,Li L,et al.Molecular cloning and differential expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Hypophthalmichthys molitrix[J].Fish Shellfish Immunol,2011,30(2):473-479.
[9]Kim K Y,Sang Y L,Cho Y S,et al.Molecular characterization and mRNA expression during metal exposure and thermal stress of copper/zinc-and manganese-superoxide dismutases in disk abalone,Haliotis discus discus[J].Fish Shellfish Immunol,2007,23(5):1043-1059.
[10]Yang J,Dong S,Jiang Q,et al.Characterization and expression of cytoplasmic copper/zinc superoxide dismutase(Cu Zn SOD)gene under temperature and hydrogen peroxide(H2O2)in rotifer Brachionus calyciflorus[J].Gene,2013,518(2):388-396.
[11]Dalziel J E,Shyan W S,Thai P,et al.Expression of human BK ion channels in Sf9 cells,their purification using metal affinity chromatography,and functional reconstitution into planar lipid bilayers[J].J Chromatogr B,2007,857(2):315-321.
[12]Santovito G,Cassini A,Piccinni E.Cu,Zn superoxide dismutase from Trematomus bernacchii:Functional conservation and erratic molecular evolution in Antarctic teleosts[J].Comp Biochem Physiol Part C Toxicol Pharmacol,2006,143(143):444-454.
[13]Ken C F,Lin C T,Shaw J F,et al.Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress[J].Mar Biotechnol,2003,5(2):167-173.
[14]Lin C T,Lee T L,Duan K J,et al.Molecular cloning,characterization,and expression of a c DNA coding Copper/Zinc superoxide dismutase from black porgy[J].J Agric Food Chem,2000,48(9):4444-4447.
[15]Li Z,Liang H W,Li L,et al.Molecular cloning and differential expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Hypophthalmichthys molitrix[J].Fish Shellfish Immunol,2011,30(2):473-479.
[16]Tian J,Sun B,Luo Y,et al.Distribution of Ig M,Ig D and Ig Z in mandarin fish,Siniperca chuatsi,lymphoid tissues and their transcriptional changes after Flavobacterium columnare,stimulation[J].Aquaculture,2009,288(1):14-21.
[17]Wang G,Li J,Zou P,et al.Expression pattern,promoter activity and bactericidal property ofβ-defensin from the mandarin fish Siniperca chuatsi[J].Fish Shellfish Immunol,2012,33(3):522-531.
[18]Sun B J,Wang G L,Xie H X,et al.Gene structure of goosetype lysozyme in the mandarin fish Siniperca chuatsi,with analysis on the lytic activity of its recombinant in Escherichia coli[J].Aquaculture,2006,252(2–4):106-113.
[19]Xiao J,Zhou Z C,Chen C,et al.Tumor necrosis factor-alpha gene from mandarin fish,Siniperca chuatsi:Molecular cloning,cytotoxicity analysis and expression profile[J].Mol Immunol,2007,44(14):3615-3622.
[20]程周玉,文春根,胡向萍,等.翘嘴鳜铜锌超氧化物歧化酶基因的分子克隆与原核表达[J].南昌大学学报(理科版),2015,39(6):587-596.
[21]Xu H H,Ma H,Hu B Q,et al.Molecular cloning,identification and functional characterization of a novel intracellular Cu-Zn superoxide dismutase from the freshwater mussel Cristaria plicata[J].Fish Shellfish Immunol,2010,29(4):615-622.
[22]Bao Y,Li L,Xu F,et al.Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians:Its gene structure,mRNA expression and recombinant protein[J].Fish Shellfish Immunol,2009,27(2):210-220.
[23]刘建荣,赵晓瑜,宋小青,等.重组人Cu,Zn-SOD包含体的复性、纯化及复性蛋白稳定性研究[J].中国药学杂志,2007,42(13):969-974.
[24]龙建银,王会信.外源基因在大肠杆菌中表达的研究进展[J].生物化学与生物物理进展,1997(2):126-132.
[25]Dalziel J E,Shyan W S,Thai P,et al.Expression of human BK ion channels in Sf9 cells,their purification using metal affinity chromatography,and functional reconstitution into planar lipid bilayers[J].J Chromatogr B,2007,857(2):315-321.
[26]田春美,钟秋平.超氧化物歧化酶的现状研究进展[J].中国热带医学,2005,5(8):1730-1732.
[27]吴燕燕,李来好,郝志明,等.罗非鱼肝脏中超氧化物歧化酶的提取、纯化与分析[J].水产学报,2007,31(4):518-524.
[28]张尔贤,李建喜.鲨鱼肝超氧化物歧化酶的纯化与部分性质研究[J].中国药学杂志,1999(4):231-233.
[29]梅光泉,廊惠芳.超氧化物歧化酶中微量元素的化学行为和生物学功效[J].微量元素与健康研究,2003,20(5):59-62.
[30]Salin M L,Oesterhelt D.Purification of a manganese-containing superoxide dismutase from Halobacterium halobium[J].Arch Biochem Biophys,1988,260(2):806-810.
[31]王伟伟,刘存岐,李道季.3种虾超氧化物歧化酶部分理化性质比较[J].水产科学,2009,28(4):200-204.