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| 人谷胱甘肽硫转移酶的克隆表达及活性鉴定 | |
| 引用本文： | 柴晓鹃,胡海红,余露山,曾苏. 人谷胱甘肽硫转移酶的克隆表达及活性鉴定[J]. 浙江大学学报(医学版), 2014, 43(2): 168-174. DOI: 10.3785/j.issn.1008-9292.2014.03.009 |
| 作者姓名： | 柴晓鹃 胡海红 余露山 曾苏 |
| 作者单位： | 浙江大学药学院药物分析与药物代谢研究室 浙江省抗肿瘤药物临床前研究重点实验室， 浙江 杭州 310058 |
| 基金项目： | 国家重大新药创制科技专项（2012ZX09506001-004）. |
| 摘 要： | 目的：克隆表达人谷胱甘肽硫转移酶GSTA1、GSTT1和GSTP1，用于研究化合物的代谢途径。方法：采用反转录PCR得到人谷胱甘肽硫转移酶GSTA1、GSTT1和GSTP1基因全长cDNA。将测序正确的cDNA连接到pET-28a阳性表达载体上并在大肠杆菌BL21(DE3)中稳定表达。利用亲和色谱纯化表达得到重组酶，以2，4-二硝基氯苯为底物，测定340 nm 波长处吸收度的变化，检测酶活性。结果：PCR产物与克隆载体pMD19-T连接后的质粒测序结果表明，目的基因的cDNA序列完全正确。表达质粒在大肠杆菌BL21(DE3)中获得良好的可溶性表达，经镍离子亲和柱纯化后目的蛋白较纯，具有较好的催化活性。所表达的hGSTA1、hGSTT1和hGSTP1酶比活性分别为17.55、0.02、18.75 μmol·min-1·mg-1蛋白。结论：成功构建了人谷胱甘肽硫转移酶GSTA1、GSTT1和GSTP1的原核表达系统，得到的重组酶可用于药物代谢研究。 |
| 关 键 词： | 谷胱甘肽转移酶/生物合成 克隆 分子 基因表达 色谱法 亲和 基因扩增 质粒 逆转录聚合酶链反应 |
| 收稿时间： | 2013-11-06 |
Expression of human glutathione S-transferase A1, P1 and T1 in Escherichia coli |
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| CHAI Xiao-juan,HU Hai-hong,YU Lu-shan,ZENG Su. Expression of human glutathione S-transferase A1, P1 and T1 in Escherichia coli[J]. Journal of Zhejiang University. Medical sciences, 2014, 43(2): 168-174. DOI: 10.3785/j.issn.1008-9292.2014.03.009 | |
| Authors: | CHAI Xiao-juan HU Hai-hong YU Lu-shan ZENG Su |
| Affiliation: | Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University; Zhejiang Provincial Key Laboratory of Anti-Cancer Drug Research, Hangzhou 310058, China |
| Abstract: | Objective： To construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli). Methods： Human GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors. The proteins were expressed in E. coli BL21(DE3). After purified by Ni2+ affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate.Results： The correct GSTA1, GSTP1 and GSTT1 genes were cloned. And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli. After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities, which were 17.55, 0.02, 18.75 μmol·min-1·mg-1, respectively. Conclusion： The expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully. |
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