人谷氧还蛋白基因的分子克隆及表达
张春晶,于海涛,齐齐哈尔医学院生化教研室 黑龙江省齐齐哈尔市 161042
周宏博,邹朝霞,董钦, 哈尔滨医科大学生化教研室 黑龙江省哈尔滨市 150086
张春晶,女,1973年生, 黑龙江省齐齐哈尔人, 硕士,主要从事生物化学与分子生物学方面的研究.
黑龙江省自然基金资助项目,No. D2004-05
通讯作者:周宏博,150086, 黑龙江省哈尔滨市南岗区保健路157号,哈尔滨医科大学生化教研室. zhouhongbo6704@yahoo.com.cn
, 百拇医药
电话: 0451-86671684
收稿日期:2005-04-15 接受日期:2005-05-06
Molecular cloning and expression of humanglutaredoxin gene
Chun-Jing Zhang, Hong-Bo Zhou, Chao-Xia Zou, Qin Dong, Hai-Tao Yu
Chun-Jing Zhang,Hai-Tao Yu, Department of Biochemistry, Qiqihar Medical College,Qiqihar 161042, Heilongjiang Province, China
, 百拇医药
Hong-Bo Zhou, Chao-Xia Zou, Qin Dong, Department of Biochemistry,Harbin Medical University, Harbin 150086, Heilongjiang Province, China
Supported by the Natural Science Foundation of HeilongjiangProvince, No.D2004-05
Correspondence to: Hong-Bo Zhou, Department of Biochemistry, HarbinMedical University, 157 Baojian Road, Nangang District, Harbin 150086,Heilongjiang Province, China. zhouhongbo6704@yahoo.com.cn
, 百拇医药
Received: 2005-04-15 Accepted:2005-05-06
Abstract
AIM: To clone and sequence glutaredoxin (Grx) cDNA from humanumbilical endothelium cells, and to express it in E.coli BL21(DE3).
METHODS: The total RNA was extracted from human umbilicalendothelium cells. Grx cDNA was obtained by reverse transcriptionpolymerase chain reaction (RT-PCR), and then cloned into pRSETA vector toconstruct recombinant pRSET-Grx. The products were transformed into E.coli(JM109). The positive transformant was identified bybacterium-specific PCR and digestion of restriction endonucleases. Thepositive clones were purified and sequenced, then transfected into E.coliBL21 (DE3). The expression of Grx was induced by IPTG.
, 百拇医药
RESULTS: Compared the cDNA sequence we obtained with that inGenBank (NM002064), there was one difference in the base pairs, but theamino acid sequence was identical. The recombinant Grx was expressed afterinduced by IPTG for 4-5 hours, and the apparent Mr16000were confirmed by 150 g/L SDS-PAGE.
CONCLUSION: Recombinant human Grx gene was successfully cloned andexpressed in E.coli.
, 百拇医药
Key Words: Molecular cloning; Glutaredoxin; Human umbilicalendothelium cells
Zhang CJ, Zhou HB, Zou CX, Dong Q, Yu HT. Molecular cloning and expressionof human glutaredoxin gene. Shijie Huaren Xiaohua Zazhi 2005;13(13):1558-1561
摘要目的:克隆人脐静脉内皮细胞谷氧还蛋白(glutaredoxin,Grx)编码区的cDNA序列并进行序列测定，构建原核表达载体并在大肠杆菌BL21(DE3)中表达.
, 百拇医药 方法:从人脐静脉内皮细胞中提取总RNA，采用RT-PCR技术，获得该基因编码区的cDNA，并重组入原核克隆表达载体pRSETA，构建重组质粒pRSET-Grx，通过菌落PCR筛选及限制性内切酶鉴定，选择阳性克隆并测序.将测序正确的重组质粒pRSET-Grx转化大肠杆菌BL21(DE3)，用IPTG诱导表达.
结果:将所得序列与GenBank提供的序列(NM002064)比较，测出的序列在核苷酸序列上有一处碱基不同，但在氨基酸序列上与已知序列一致.经IPTG诱导4-5 h后，150 g/L SDS-PAGE 分析，表达出Mr16000的蛋白.
结论:从人脐静脉内皮细胞中成功地获得Grx编码区的cDNA，成功构建了原核融合表达载体pRSET-Grx并获得表达.
, 百拇医药
关键词:分子克隆; 谷氧还蛋白;人脐静脉内皮细胞
张春晶,周宏博,邹朝霞, 董钦,于海涛.人谷氧还蛋白基因的分子克隆及表达.世界华人消化杂志 2005;13(13):1558-15611 (PDF)总RNA提取.
图2 (PDF) Grx 基因的PCR扩增.1-3: PCR product; 4: PCR Marker (1057，770，612，495，392，341，297，210bp fragment，fromtop to bottom).
图3 (PDF) 重组质粒pRSET-Grx酶切鉴定.1: PCR Marker; 2: pRSET-Grx digested with BamHⅠ+EcoRⅠ;3: undigested pRSET-Grx; 4: undigested pRSETA; 5: pRSETA digested with BamHⅠ+EcoRⅠ;6: plasmid Marker.
, 百拇医药
2.1 人脐静脉内皮细胞Grx基因序列测定 测定的基因序列长370bp，ORF为320bp，部分序列测序图谱结果见图4.用Dnasis和Pmsis软件程序与GenBankData Base中发表的人Grx基因序列进行同源性分析比较，同源性为99.7%，碱基序列只有1处不同，即240T-C(前者为人皮肤细胞Grx基因)，但相应氨基酸序列(Ile)没有发生改变.
图4 (PDF) 人脐静脉内皮细胞Grx编码区的cDNA序列.
2.2 重组pRSET-GRX质粒在大肠杆菌中的表达将筛选的重组克隆子转化大肠杆菌BL21(DE3)，获得重组表达菌株，经0.5mmol/LIPTG，37℃诱导培养4-5h，其150 g/L SDS-PAGE结果(图5)可见，表达产物的Mr16000.由于人的谷氧还蛋白含107个氨基酸，Mr11800，由于pRSET-Grx表达载体5’端带有用于表达蛋白纯化的多聚His标签，维持目的基因稳定转录的T7噬菌体基因10起动子、用于表达抗体检测的抗原决定基因及肠激酶裂解识别位点等标记，因此谷氧还蛋白的融合蛋白的Mr15840，SDS-PAGE结果与理论值相符.
, 百拇医药
图5 (PDF) 150 g/L SDS-PAGE分析表达产物. 1: Bacterial sample before induced; 2: Bacterial sample afterinduced 4 h; 3: Bacterial sample after induced 5 h; 4: Low-range proteinmolecular weight markers.
3 讨论由于血管内皮细胞具有复杂的生物学功能，在国内外医学界逐渐引起重视，目前研究最多的是脐静脉内皮细胞.血管内皮细胞具有活跃的分泌与代谢功能，他在调节血管功能、维持心血管生理稳态中起着重要作用.血管内皮也是多种心血管疾病或危险因素作用的重要靶器官，组成人体的脐静脉内皮细胞除受到内源性氧化应激损伤外，还受到血液系统中的氧化性物质的作用.血管内皮细胞功能障碍时，导致NO分泌异常，与高血压、高血脂症、缺血再灌注的损害和动脉粥样硬化等许多心血管疾病的发生发展有密切关系[10].
, http://www.100md.com
有文献报道，在活性氧引起的损伤中，蛋白质氧化损伤先于核酸[11]，蛋白发生羰基化和糖基化，从而失去生物活性.已有研究证明，关于蛋白活性的修复，除机体内小分子物质发挥作用外，主要依赖于巯基-二硫键氧化还原酶家族，而Grx是这个家族的重要组分[12]，他是利用GSH作为辅酶来催化体内氧化状态的蛋白质上的二硫键还原为巯基，恢复蛋白质结构和功能，修复蛋白质活性的抗氧化酶，对维持体内稳定的氧化还原状态及对活性氧所导致的氧化应激损伤有阻抗和治疗作用[13].最近研究发现，Grx是机体内能特异、高效的还原谷胱甘肽化蛋白的一种酶蛋白[14-15]，Grx特异的恢复氧化应激损伤产生的谷胱甘肽化蛋白活性的能力可能会使其成为热点药物.目前，细胞内Grx抵御过氧化氢作用的研究国外已有很多报道[6-9]，但是关于Grx本身抵御过氧化氢作用的研究，至今未见报道.因此，该蛋白的原核克隆、高效表达及观察Grx保护细胞免受氧化应激损伤作用的研究，将对于人类氧化应激相关疾病的预防和治疗有重要的意义.目前，根据本研究的实验结果，我们正在进行Grx的优化表达、纯化及体外抗氧化活性的研究，以便为Grx生物学功能研究及今后的临床应用提供重要的理论依据.另外，本实验经30个PCR循环，370bp的基因序列中有一处碱基与已知序列的不同，是PCR过程中的错误参入还是人脐静脉内皮细胞中Grx编码区的cDNA序列就是如此，还有待进一步论证.但本实验中出现的不同碱基没有影响氨基酸的排序，因此，即使是错配也不必去做更正.
, 百拇医药
4 参考文献1 Holmgren A. Hydrogen donor system for Escherichia coliribonucleoside-diphosphate reductase dependent upon
glutathione. Proc Natl Acad Sci USA 1976;73:2275-2279
2 Daily D, Vlamis-Gardikas A, Offen D, Mittelman L, Melamed E,Holmgren A, Barzilai A. Glutaredoxin
protects cerebellar granule neurons fromdopamine-induced apoptosis by activating NF-kappa B via Ref-1.
, http://www.100md.com
J Biol Chem 2001;276:1335-1344
3 Song JJ, Rhee JG, Suntharalingam M, Walsh SA, Spitz DR, Lee YJ.Role of glutaredoxin in metabolic oxidative stress.
Glutaredoxin as a sensor of oxidativestress mediated by H2O2. JBiol Chem 2002;277:46566-46575
4 Landino LM, Moynihan KL, Todd JV, Kennett KL. Modulation of theredox state of tubulin by the glutathione/glutaredoxin
, 百拇医药
reductase system. Biochem Biophys ResCommun 2004;314:555-560
5 Bandyopadhyay S, Starke DW, Mieyal JJ, Gronostajski RM.Thioltransferase (glutaredoxin) reactivates the DNA-binding
activity of oxidation-inactivated nuclearfactor I. J Biol Chem 1998;273:392-397
6 Prieto-Alamo MJ, Jurado J, Gallardo-Madueno R, Monje-Casas F,Holmgren A, Pueyo C. Transcriptional
, 百拇医药 regulation of glutaredoxin and thioredoxinpathways and related enzymes in response to oxidative stress.
J Biol Chem 2000;275:13398-13405
7 Ragbavacbari N, Krysan K, Xing K, Loum F. Regulation ofthioltransferase expression in human lens epithelial cells.
Investg Ophthalmol Vis Sci 2001;42:1002-1008
8 Okuda M, Inoue N, Azumi H, Seno T, Sumi Y, Hirata Ki, Kawashima S,Hayashi Y, Itoh H, Yodoi J, Yokoyama M.
, 百拇医药
Expression of glutaredoxin in humancoronary arteries: its potential role in antioxidant protection againstatherosclerosis.
Arterioscler Thromb Vasc Biol 2001;21:1483-1487
9 Sahlin L, Wang H, Stjernholm Y, Lundberg M, Ekman G, Holmgren A,Eriksson H. The expression of glutaredoxin is
increased in the human cervix in termpregnancy and immediately post-partum, particularly afterprostaglandin-induced
, 百拇医药
delivery. Mol Hum Reprod 2000;6:1147-1153
10 Schwartz EA, Bizios R, Medow MS, Gerritsen ME. Exposure of humanvascular endothelial cells to sustained hydrostatic
pressure stimulates proliferation.Involvement of the alphaV integrins. Circ Res 1999;84:315-322
11 陈媛, 周玫.自由基医学基础与病理生理.第一版.北京: 人民卫生出版社, 2002:14-35
12 Jung CH, Thomas JA. S-glutathiolated hepatocyte proteins andinsulin disulfides as substrates for reduction by
, 百拇医药
glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione.Arch Biochem Biophys 1996;335:61-72
13 Holmgren A. Antioxidant function of thiodoxin and glutaredoxinsystems. Antioxid Redox Signal 2000;2:811-820
14 Rebrin I, Kamzalov S, Sohal RS. Effects of age and caloricrestriction on glutathione redox state in mice.
Free Radic Biol Med 2003;35:626-635
15 Yoshitake S, Nanri H, Fernando MR, Minakami S. Possible differencesin the regenerative roles played by
thioltransferase and thioredoxin for oxidativeely damaged proteins. JBiochem (Tokyo) 1994;116:42-46
编辑 潘伯荣 审读 张海宁, http://www.100md.com( 张春晶, 周宏博, 邹朝霞, 董 钦, 于海涛)
周宏博,邹朝霞,董钦, 哈尔滨医科大学生化教研室 黑龙江省哈尔滨市 150086
张春晶,女,1973年生, 黑龙江省齐齐哈尔人, 硕士,主要从事生物化学与分子生物学方面的研究.
黑龙江省自然基金资助项目,No. D2004-05
通讯作者:周宏博,150086, 黑龙江省哈尔滨市南岗区保健路157号,哈尔滨医科大学生化教研室. zhouhongbo6704@yahoo.com.cn
, 百拇医药
电话: 0451-86671684
收稿日期:2005-04-15 接受日期:2005-05-06
Molecular cloning and expression of humanglutaredoxin gene
Chun-Jing Zhang, Hong-Bo Zhou, Chao-Xia Zou, Qin Dong, Hai-Tao Yu
Chun-Jing Zhang,Hai-Tao Yu, Department of Biochemistry, Qiqihar Medical College,Qiqihar 161042, Heilongjiang Province, China
, 百拇医药
Hong-Bo Zhou, Chao-Xia Zou, Qin Dong, Department of Biochemistry,Harbin Medical University, Harbin 150086, Heilongjiang Province, China
Supported by the Natural Science Foundation of HeilongjiangProvince, No.D2004-05
Correspondence to: Hong-Bo Zhou, Department of Biochemistry, HarbinMedical University, 157 Baojian Road, Nangang District, Harbin 150086,Heilongjiang Province, China. zhouhongbo6704@yahoo.com.cn
, 百拇医药
Received: 2005-04-15 Accepted:2005-05-06
Abstract
AIM: To clone and sequence glutaredoxin (Grx) cDNA from humanumbilical endothelium cells, and to express it in E.coli BL21(DE3).
METHODS: The total RNA was extracted from human umbilicalendothelium cells. Grx cDNA was obtained by reverse transcriptionpolymerase chain reaction (RT-PCR), and then cloned into pRSETA vector toconstruct recombinant pRSET-Grx. The products were transformed into E.coli(JM109). The positive transformant was identified bybacterium-specific PCR and digestion of restriction endonucleases. Thepositive clones were purified and sequenced, then transfected into E.coliBL21 (DE3). The expression of Grx was induced by IPTG.
, 百拇医药
RESULTS: Compared the cDNA sequence we obtained with that inGenBank (NM002064), there was one difference in the base pairs, but theamino acid sequence was identical. The recombinant Grx was expressed afterinduced by IPTG for 4-5 hours, and the apparent Mr16000were confirmed by 150 g/L SDS-PAGE.
CONCLUSION: Recombinant human Grx gene was successfully cloned andexpressed in E.coli.
, 百拇医药
Key Words: Molecular cloning; Glutaredoxin; Human umbilicalendothelium cells
Zhang CJ, Zhou HB, Zou CX, Dong Q, Yu HT. Molecular cloning and expressionof human glutaredoxin gene. Shijie Huaren Xiaohua Zazhi 2005;13(13):1558-1561
摘要目的:克隆人脐静脉内皮细胞谷氧还蛋白(glutaredoxin,Grx)编码区的cDNA序列并进行序列测定，构建原核表达载体并在大肠杆菌BL21(DE3)中表达.
, 百拇医药 方法:从人脐静脉内皮细胞中提取总RNA，采用RT-PCR技术，获得该基因编码区的cDNA，并重组入原核克隆表达载体pRSETA，构建重组质粒pRSET-Grx，通过菌落PCR筛选及限制性内切酶鉴定，选择阳性克隆并测序.将测序正确的重组质粒pRSET-Grx转化大肠杆菌BL21(DE3)，用IPTG诱导表达.
结果:将所得序列与GenBank提供的序列(NM002064)比较，测出的序列在核苷酸序列上有一处碱基不同，但在氨基酸序列上与已知序列一致.经IPTG诱导4-5 h后，150 g/L SDS-PAGE 分析，表达出Mr16000的蛋白.
结论:从人脐静脉内皮细胞中成功地获得Grx编码区的cDNA，成功构建了原核融合表达载体pRSET-Grx并获得表达.
, 百拇医药
关键词:分子克隆; 谷氧还蛋白;人脐静脉内皮细胞
张春晶,周宏博,邹朝霞, 董钦,于海涛.人谷氧还蛋白基因的分子克隆及表达.世界华人消化杂志 2005;13(13):1558-15611 (PDF)总RNA提取.
图2 (PDF) Grx 基因的PCR扩增.1-3: PCR product; 4: PCR Marker (1057，770，612，495，392，341，297，210bp fragment，fromtop to bottom).
图3 (PDF) 重组质粒pRSET-Grx酶切鉴定.1: PCR Marker; 2: pRSET-Grx digested with BamHⅠ+EcoRⅠ;3: undigested pRSET-Grx; 4: undigested pRSETA; 5: pRSETA digested with BamHⅠ+EcoRⅠ;6: plasmid Marker.
, 百拇医药
2.1 人脐静脉内皮细胞Grx基因序列测定 测定的基因序列长370bp，ORF为320bp，部分序列测序图谱结果见图4.用Dnasis和Pmsis软件程序与GenBankData Base中发表的人Grx基因序列进行同源性分析比较，同源性为99.7%，碱基序列只有1处不同，即240T-C(前者为人皮肤细胞Grx基因)，但相应氨基酸序列(Ile)没有发生改变.
图4 (PDF) 人脐静脉内皮细胞Grx编码区的cDNA序列.
2.2 重组pRSET-GRX质粒在大肠杆菌中的表达将筛选的重组克隆子转化大肠杆菌BL21(DE3)，获得重组表达菌株，经0.5mmol/LIPTG，37℃诱导培养4-5h，其150 g/L SDS-PAGE结果(图5)可见，表达产物的Mr16000.由于人的谷氧还蛋白含107个氨基酸，Mr11800，由于pRSET-Grx表达载体5’端带有用于表达蛋白纯化的多聚His标签，维持目的基因稳定转录的T7噬菌体基因10起动子、用于表达抗体检测的抗原决定基因及肠激酶裂解识别位点等标记，因此谷氧还蛋白的融合蛋白的Mr15840，SDS-PAGE结果与理论值相符.
, 百拇医药
图5 (PDF) 150 g/L SDS-PAGE分析表达产物. 1: Bacterial sample before induced; 2: Bacterial sample afterinduced 4 h; 3: Bacterial sample after induced 5 h; 4: Low-range proteinmolecular weight markers.
3 讨论由于血管内皮细胞具有复杂的生物学功能，在国内外医学界逐渐引起重视，目前研究最多的是脐静脉内皮细胞.血管内皮细胞具有活跃的分泌与代谢功能，他在调节血管功能、维持心血管生理稳态中起着重要作用.血管内皮也是多种心血管疾病或危险因素作用的重要靶器官，组成人体的脐静脉内皮细胞除受到内源性氧化应激损伤外，还受到血液系统中的氧化性物质的作用.血管内皮细胞功能障碍时，导致NO分泌异常，与高血压、高血脂症、缺血再灌注的损害和动脉粥样硬化等许多心血管疾病的发生发展有密切关系[10].
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有文献报道，在活性氧引起的损伤中，蛋白质氧化损伤先于核酸[11]，蛋白发生羰基化和糖基化，从而失去生物活性.已有研究证明，关于蛋白活性的修复，除机体内小分子物质发挥作用外，主要依赖于巯基-二硫键氧化还原酶家族，而Grx是这个家族的重要组分[12]，他是利用GSH作为辅酶来催化体内氧化状态的蛋白质上的二硫键还原为巯基，恢复蛋白质结构和功能，修复蛋白质活性的抗氧化酶，对维持体内稳定的氧化还原状态及对活性氧所导致的氧化应激损伤有阻抗和治疗作用[13].最近研究发现，Grx是机体内能特异、高效的还原谷胱甘肽化蛋白的一种酶蛋白[14-15]，Grx特异的恢复氧化应激损伤产生的谷胱甘肽化蛋白活性的能力可能会使其成为热点药物.目前，细胞内Grx抵御过氧化氢作用的研究国外已有很多报道[6-9]，但是关于Grx本身抵御过氧化氢作用的研究，至今未见报道.因此，该蛋白的原核克隆、高效表达及观察Grx保护细胞免受氧化应激损伤作用的研究，将对于人类氧化应激相关疾病的预防和治疗有重要的意义.目前，根据本研究的实验结果，我们正在进行Grx的优化表达、纯化及体外抗氧化活性的研究，以便为Grx生物学功能研究及今后的临床应用提供重要的理论依据.另外，本实验经30个PCR循环，370bp的基因序列中有一处碱基与已知序列的不同，是PCR过程中的错误参入还是人脐静脉内皮细胞中Grx编码区的cDNA序列就是如此，还有待进一步论证.但本实验中出现的不同碱基没有影响氨基酸的排序，因此，即使是错配也不必去做更正.
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