人UGT1A6重組酶的表達(dá)與化合物葡醛酸結(jié)合研究
發(fā)布時(shí)間：2018-11-08 16:19
【摘要】：尿苷二磷酸葡醛酸轉(zhuǎn)移酶(uridine 5'diphosphate glucuronosyltransferases,UGTs)是一類Ⅱ相代謝酶,能催化葡醛酸與其底物結(jié)合。該過程是機(jī)體的重要排泄途徑之一。過去,藥物代謝評(píng)價(jià)常用組織微粒體進(jìn)行研究,但組織微粒體中酶系復(fù)雜,且UGT酶系屬于超基因家族酶,各同工酶的底物特異性存在著交叉重疊。由于缺乏各同工酶的特異性抑制劑,所以鑒定各UGT同工酶的底物比較復(fù)雜。目前,人們已開始廣泛采用體外克隆并表達(dá)人體藥物代謝酶來進(jìn)行藥物代謝研究,且大多數(shù)UGT同工酶已通過不同表達(dá)方式在體外獲得表達(dá)。例如本實(shí)驗(yàn)室已在CHO、CHL、V79等哺乳動(dòng)物細(xì)胞中成功表達(dá)人UGT1A3、1A9。近來,利用桿狀病毒-昆蟲表達(dá)系統(tǒng)表達(dá)外源基因的技術(shù)也得到廣泛地應(yīng)用,本課題通過該系統(tǒng)來表達(dá)人UGT1亞族成員之一UGT1A6酶,并用該重組酶對(duì)一些化合物進(jìn)行葡醛酸結(jié)合研究。在明確基因構(gòu)成的情況下獲得由特定基因所表達(dá)的UGT同工酶可以保證酶的單一性,因此能針對(duì)性地進(jìn)行其代謝功能研究,了解化合物Ⅱ相葡醛酸化情況,預(yù)測(cè)所研究的化合物可能帶來的體內(nèi)葡醛酸化程度及化合物之間的相互作用。 1 UGT1A6基因的克隆與重組病毒的構(gòu)建 采用RT-PCR技術(shù)從人肝組織中擴(kuò)增出UGT1A6基因的cDNA,通過克隆載體pGEM-T擴(kuò)增后,經(jīng)HindⅢ/XhoⅠ雙酶切得到帶粘性末端的UGT1A6基因片段與表達(dá)載體pcDNA3.1(+)定向連接,亞克隆后經(jīng)BSP TI/XhoⅠ雙酶切處理,電
[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，
本文編號(hào)：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，
本文編號(hào)：2319016
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