人UGT1A6重组酶的表达与化合物葡醛酸结合研究
发布时间：2018-11-08 16:19
【摘要】：尿苷二磷酸葡醛酸转移酶(uridine 5'diphosphate glucuronosyltransferases,UGTs)是一类Ⅱ相代谢酶,能催化葡醛酸与其底物结合。该过程是机体的重要排泄途径之一。过去,药物代谢评价常用组织微粒体进行研究,但组织微粒体中酶系复杂,且UGT酶系属于超基因家族酶,各同工酶的底物特异性存在着交叉重叠。由于缺乏各同工酶的特异性抑制剂,所以鉴定各UGT同工酶的底物比较复杂。目前,人们已开始广泛采用体外克隆并表达人体药物代谢酶来进行药物代谢研究,且大多数UGT同工酶已通过不同表达方式在体外获得表达。例如本实验室已在CHO、CHL、V79等哺乳动物细胞中成功表达人UGT1A3、1A9。近来,利用杆状病毒-昆虫表达系统表达外源基因的技术也得到广泛地应用,本课题通过该系统来表达人UGT1亚族成员之一UGT1A6酶,并用该重组酶对一些化合物进行葡醛酸结合研究。在明确基因构成的情况下获得由特定基因所表达的UGT同工酶可以保证酶的单一性,因此能针对性地进行其代谢功能研究,了解化合物Ⅱ相葡醛酸化情况,预测所研究的化合物可能带来的体内葡醛酸化程度及化合物之间的相互作用。 1 UGT1A6基因的克隆与重组病毒的构建 采用RT-PCR技术从人肝组织中扩增出UGT1A6基因的cDNA,通过克隆载体pGEM-T扩增后,经HindⅢ/XhoⅠ双酶切得到带粘性末端的UGT1A6基因片段与表达载体pcDNA3.1(+)定向连接,亚克隆后经BSP TI/XhoⅠ双酶切处理,电
[Abstract]:Uridine diphosphate glucuronyltransferase (uridine 5'diphosphate glucuronosyltransferases,UGTs) is a kind of phase II metabolic enzyme which can catalyze the binding of uronic acid to its substrate. This process is one of the important excretion pathways. In the past, tissue microsomes were commonly used to evaluate drug metabolism, but the enzyme system in tissue microsomes was complex, and the enzyme system of UGT belonged to supergene family, and the substrate specificity of each isozyme was overlapping. Due to the lack of specific inhibitors of each isozyme, it is complicated to identify the substrates of each UGT isozyme. At present, human drug metabolism enzymes have been extensively cloned and expressed in vitro, and most of the UGT isozymes have been expressed in vitro. For example, our laboratory has successfully expressed human UGT1A3,1A9. in mammalian cells such as CHO,CHL,V79. Recently, the technique of expressing exogenous genes by baculovirus-insect expression system has been widely used. In this study, UGT1A6 enzyme, one of the members of human UGT1 subfamily, was expressed by this system. Some compounds were studied by glucuronic acid binding with the recombinant enzyme. The UGT isozyme expressed by a specific gene can guarantee the singularity of the enzyme, so the metabolic function of the isozyme can be studied in order to understand the status of glucuronation in compound 鈪，
本文编号：2319016
[Abstract]:Uridine diphosphate glucuronyltransferase (uridine 5'diphosphate glucuronosyltransferases,UGTs) is a kind of phase II metabolic enzyme which can catalyze the binding of uronic acid to its substrate. This process is one of the important excretion pathways. In the past, tissue microsomes were commonly used to evaluate drug metabolism, but the enzyme system in tissue microsomes was complex, and the enzyme system of UGT belonged to supergene family, and the substrate specificity of each isozyme was overlapping. Due to the lack of specific inhibitors of each isozyme, it is complicated to identify the substrates of each UGT isozyme. At present, human drug metabolism enzymes have been extensively cloned and expressed in vitro, and most of the UGT isozymes have been expressed in vitro. For example, our laboratory has successfully expressed human UGT1A3,1A9. in mammalian cells such as CHO,CHL,V79. Recently, the technique of expressing exogenous genes by baculovirus-insect expression system has been widely used. In this study, UGT1A6 enzyme, one of the members of human UGT1 subfamily, was expressed by this system. Some compounds were studied by glucuronic acid binding with the recombinant enzyme. The UGT isozyme expressed by a specific gene can guarantee the singularity of the enzyme, so the metabolic function of the isozyme can be studied in order to understand the status of glucuronation in compound 鈪，
本文编号：2319016
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