黄瓜Cu/Zn-SOD基因克隆及可溶性表达和纯化
|
摘要
[目的]克隆黄瓜Cu/Zn-SOD基因,并利用大肠杆菌进行可溶性表达、纯化及活性测定。[方法]采用Trizol法提取黄瓜表皮的总RNA,然后设计特异性引物,通过RT-PCR技术克隆获得黄瓜Cu/Zn-SOD基因。该基因与p GEX2T载体相连后,转化大肠杆菌BL21(DE3),摸索可溶性表达方法。利用GST亲和层析方法纯化目标蛋白,SOD酶活性测定采用邻苯三酚法。[结果]成功地克隆了全长为459 bp的黄瓜Cu/Zn-SOD基因,编码152个氨基酸。Protein Blast分析表明其结构域中分别包含Cu2+、Zn2+结合位点,为典型的Cu/Zn-SOD酶。目标蛋白可溶性表达条件是16℃、0. 1 mmol/L IPTG诱导20 h。SDS-PAGE分析表明GST亲和层析成功地获得重组黄瓜Cu/Zn-SOD。活性测定表明两次纯化的蛋白样品SOD酶活力分别为2 328. 9 U/m L、2 144. 7 U/m L。[结论]从黄瓜表皮中克隆获得Cu/Zn-SOD基因,该基因在大肠杆菌系统中获得可溶性表达,纯化后的重组蛋白具有较高的SOD酶活力。
[Objective]To clone the Cu/Zn-SOD gene of cucumber,and explore soluble expression of Cu/Zn-SOD gene in E. coli,then purify the target protein and determinate the activity of recombinant Cu/Zn-SOD. [Method]Total RNA was extracted from cucumber epidermis by Trizol method. The specific primers were designed the Cu/Zn-SOD gene of cucumber was cloned by RT-PCR technology. The target gene was inserted into p GEX2 T plasmid,then recombinant plasmid was transformed into E. coli BL21( DE3) and explore the soluble expression method. The target protein was purified by GST affinity method,and pyrogallol autoxidation method was used to determine the activity of recombinant SOD.[Result]Cu/Zn-SOD gene of cucumber,which was 459 bp,was successfully cloned. Protein blast analysis showed that it`s domain contained Cu2 +and Zn2 +binding site,and belonged to typical Cu/Zn-SOD enzyme. Soluble expression of target protein was also induced in condition of 20 h at16 ℃ and 0. 1 mmol/L IPTG. Recombinant Cu/Zn-SOD of cucumber was purified by GST affinity method and the activity of first elution protein was 2 328. 9 U/m L and second elution was 2 144. 7 U/m L. [Conclusion]Cu/Zn-SOD gene was cloned from cucumber epidermis and soluble expression of the gene in E. coli system; The purified recombinant protein has high SOD activity.
[Objective]To clone the Cu/Zn-SOD gene of cucumber,and explore soluble expression of Cu/Zn-SOD gene in E. coli,then purify the target protein and determinate the activity of recombinant Cu/Zn-SOD. [Method]Total RNA was extracted from cucumber epidermis by Trizol method. The specific primers were designed the Cu/Zn-SOD gene of cucumber was cloned by RT-PCR technology. The target gene was inserted into p GEX2 T plasmid,then recombinant plasmid was transformed into E. coli BL21( DE3) and explore the soluble expression method. The target protein was purified by GST affinity method,and pyrogallol autoxidation method was used to determine the activity of recombinant SOD.[Result]Cu/Zn-SOD gene of cucumber,which was 459 bp,was successfully cloned. Protein blast analysis showed that it`s domain contained Cu2 +and Zn2 +binding site,and belonged to typical Cu/Zn-SOD enzyme. Soluble expression of target protein was also induced in condition of 20 h at16 ℃ and 0. 1 mmol/L IPTG. Recombinant Cu/Zn-SOD of cucumber was purified by GST affinity method and the activity of first elution protein was 2 328. 9 U/m L and second elution was 2 144. 7 U/m L. [Conclusion]Cu/Zn-SOD gene was cloned from cucumber epidermis and soluble expression of the gene in E. coli system; The purified recombinant protein has high SOD activity.
引文
[1]董亮,何永志,王远亮,等.超氧化物歧化酶(SOD)的应用研究进展[J].中国农业科技导报,2013,15(5):53-58.
[2]周轩,张天,任晓敏,等.SOD提取工艺研究进展[J].粮食与食品工业,2015,22(4):57-62.
[3]牛蓓,宋君,符佳,等.乌桕Cu/Zn超氧化物歧化酶基因克隆及耐盐性[J].四川师范大学学报:自然科学版,2016,39(5):743-746.
[4]曾秀存,孙万仓,孙佳,等.白菜型冬油菜Fe-SOD基因的克隆及表达分析[J].中国农业科学,2013,46(21):4603-4611.
[5]赵怡,凌辉生,李任强.枯草芽孢杆菌Mn-SOD基因的克隆及原核表达[J].生态科学,2011,30(2):174-177.
[6]Qin Y,Dai W,Wang Y,et al.Fe-SOD cooperates with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo[J].Biochem Biophys Res Commun,2013,431(2):169-175.
[7]陈祥娥,凌沛学,张天民.超氧化物歧化酶的应用[J].食品与药品,2013,15(4):283-286.
[8]朱海生,刘建汀,陈敏氡,等.丝瓜Cu/Zn-SOD基因家族的克隆与表达分析[J].中国农业科学,2017,50(17):3386-3394.
[9]马伟荣,单春会,童军茂,等.哈密瓜铜锌超氧化物歧化酶Cu/Zn-SOD基因克隆及生物信息学分析[J].食品工业科技,2014,35(13):181-184.
[10]王岚,刘新,张帆,等.重组人铜锌超氧化物歧化酶在大肠杆菌中的表达研究[J].沈阳医学院学报,2015,17(2):70-73.
[11]Blackney MJ,Cox R,Shepherd D,et al.Cloning and expression analysis of drosophila extracellular Cu/Zn superoxide dismutase[J].Biosci.Rep,2014,34(6):851-861.
[12]Liu J,Hou J,Xia ZY,et al.Recombinant PTD-Cu/Zn SOD attenuates hypoxia-reoxygenation injury in cardiomyocytes[J].Free Radic Res,2013,47(5):386-393.
[13]Yin C,Zhao W,Zheng L,et al.High-level expression of a manganese superoxide dismutase(Po Mn-SOD)from Pleurotus ostreatus in Pichia pastoris[J].Appl Biochem Biotechnol,2014,174(1):259-269.
[14]相微微,王建斌.不同品种绿豆SOD提取工艺优化及活性的研究[J].陕西农业科学,2017,63(05):17-21.
[15]王美,武纪汝,刘艳芳,等.响应面法优化费菜SOD提取条件的研究[J].安徽农业科学,2016,44(20):63-65.
[16]付安妮,高明波,冯杰.刺梨中SOD的提取和酶活性测定[J].广州化工,2016,44(16):144-146.
[17]Che M,Wang R,Li X,et al.Expanding roles of superoxide dismutases in cell regulation and cancer[J].Drug Discov Today,2016,21(1):143-149.
[2]周轩,张天,任晓敏,等.SOD提取工艺研究进展[J].粮食与食品工业,2015,22(4):57-62.
[3]牛蓓,宋君,符佳,等.乌桕Cu/Zn超氧化物歧化酶基因克隆及耐盐性[J].四川师范大学学报:自然科学版,2016,39(5):743-746.
[4]曾秀存,孙万仓,孙佳,等.白菜型冬油菜Fe-SOD基因的克隆及表达分析[J].中国农业科学,2013,46(21):4603-4611.
[5]赵怡,凌辉生,李任强.枯草芽孢杆菌Mn-SOD基因的克隆及原核表达[J].生态科学,2011,30(2):174-177.
[6]Qin Y,Dai W,Wang Y,et al.Fe-SOD cooperates with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo[J].Biochem Biophys Res Commun,2013,431(2):169-175.
[7]陈祥娥,凌沛学,张天民.超氧化物歧化酶的应用[J].食品与药品,2013,15(4):283-286.
[8]朱海生,刘建汀,陈敏氡,等.丝瓜Cu/Zn-SOD基因家族的克隆与表达分析[J].中国农业科学,2017,50(17):3386-3394.
[9]马伟荣,单春会,童军茂,等.哈密瓜铜锌超氧化物歧化酶Cu/Zn-SOD基因克隆及生物信息学分析[J].食品工业科技,2014,35(13):181-184.
[10]王岚,刘新,张帆,等.重组人铜锌超氧化物歧化酶在大肠杆菌中的表达研究[J].沈阳医学院学报,2015,17(2):70-73.
[11]Blackney MJ,Cox R,Shepherd D,et al.Cloning and expression analysis of drosophila extracellular Cu/Zn superoxide dismutase[J].Biosci.Rep,2014,34(6):851-861.
[12]Liu J,Hou J,Xia ZY,et al.Recombinant PTD-Cu/Zn SOD attenuates hypoxia-reoxygenation injury in cardiomyocytes[J].Free Radic Res,2013,47(5):386-393.
[13]Yin C,Zhao W,Zheng L,et al.High-level expression of a manganese superoxide dismutase(Po Mn-SOD)from Pleurotus ostreatus in Pichia pastoris[J].Appl Biochem Biotechnol,2014,174(1):259-269.
[14]相微微,王建斌.不同品种绿豆SOD提取工艺优化及活性的研究[J].陕西农业科学,2017,63(05):17-21.
[15]王美,武纪汝,刘艳芳,等.响应面法优化费菜SOD提取条件的研究[J].安徽农业科学,2016,44(20):63-65.
[16]付安妮,高明波,冯杰.刺梨中SOD的提取和酶活性测定[J].广州化工,2016,44(16):144-146.
[17]Che M,Wang R,Li X,et al.Expanding roles of superoxide dismutases in cell regulation and cancer[J].Drug Discov Today,2016,21(1):143-149.