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柴晓鹃

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人谷胱甘肽硫转移酶的克隆表达及活性鉴定被引量:1
2014年
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-28 a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、18.75μmol· 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。
柴晓鹃胡海红余露山曾苏
关键词:谷胱甘肽转移酶基因扩增逆转录聚合酶链反应
Reversed-phase high-performance liquid chromatographic analysis of seven pairs of chiral drug enantiomers in transport medium after chiral derivatization
2010年
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was established for the determination of the enantiomers of 7 aryloxy aminopropanol drugs (atenolol, sotalol, celiprolol, carvedilol, metoprolol, propranolol and propafenone) in transport medium. The method involved liquid-liquid extraction of chiral drugs from transport medium, and employed 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC, 1.0 mg/mL in acetonitrile) as a pre-column chiral derivatization reagent. After derivatization, the products were separated on an Agilent Zorbax C8 column (150 min×4.6 mm, 5 μm) at 25 ℃. The mobile phase consisted of a mixture of acetonitrile and 0.01 M phosphate buffer (pH 3.5). The present methods were specific for the determination of enantiomers of each chiral drug. The absolute recoveries of the enantiomers and internal standards were 〉78%. The relative recoveries of the enantiomers were approximately 100%. The intra- and inter-day variations were 〈 15%. The method was reproducible and sufficiently sensitive to determine the enantiomers of seven aryloxy aminopropanol drugs in transport medium. The method could be used to study the transport of atenolol, sotalol, celiprolol, carvedilol, metoprolol, propranolol and propafenone.
贺瑛柴晓鹃曾苏
关键词:RP-HPLC
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