hPPARs天然配体筛选平台的建立及应用

发布时间：2017-12-28 10:58

本文关键词：hPPARs天然配体筛选平台的建立及应用　出处：《重庆医科大学》2008年博士论文　论文类型：学位论文

更多相关文章： MBP hPPARs RBA 转录激活 激动剂

【摘要】： 目的建立人过氧化物酶体增殖物活化受体(human peroxisome prolifterator-activated receptors,hPPARs)配体的筛选平台,并从中药单体中筛查hPPARs的天然配体。方法从人肝癌组织提取总RNA,RT-PCR扩增出hPPARs配体结合区(ligand binding domain,LBD)cDNA,将其克隆入表达载体pMAL-p2x麦芽糖结合蛋白(maltose binding protein,MBP)编码基因malE序列的下游,转化入E coli.TB1。含重组质粒的宿主菌经最佳优化条件诱导后,超声破碎取上清。产物用多糖亲和树脂进行纯化,纯化的MBP-hPPARsLBD用Xa因子酶切、多糖亲和树脂层析、DEAE-52阴离子交换层析纯化回收。放射性标记配基结合实验(radioligand binding assays,RBA)鉴定MBP-hPPARsLBD和hPPARsLBD的配体结合功能。高效液相色谱紫外检测法(high performance liquid chromatography and ultraviolet detection,HPLC-UV)建立hPPARsLBD配体的筛选平台,并对待试中药单体进行筛选,用经典的放射性标记配基竞争结合实验(radioligand binding competition assays,RBCA)法对HPLC-UV法筛选结果进行检验。反式激活报告基因法测定筛出药物的功能活性。结果含重组质粒的宿主菌经最佳优化条件(0.4 mmol/L,30℃振荡培养6 h)诱导,从1L培养物中可获得约20 mg MBP-hPPARsLBD,约占胞质总蛋白的31%。经纯化以及酶切回收,获得了电泳纯的MBP-hPPARsLBD和hPPARsLBD,其产量分别为14 mg/L和5 mg/L,得率分别为70%和45%。RBA显示MBP-hPPARsLBD和hPPARsLBD的结合配体功能是等效的。根据分子排阻色谱分离(Molecular Exclusion Chromatography,MEC)理论建立了HPLC-UV法hPPARs配体筛选平台。用该平台对待试药进行了筛选,结果显示小檗碱(berberine,Ber)和柚皮素(naringenin,Nar)能特异性结合hPPARα,大豆苷元(daidzein ,Dai)能特异性结合hPPARα和hPPARγ1。RCBA法结果与HPLC-UV法相同。用反式激活报告基因法测试待试中药单体的功能活性,结果显示,Dai、Ber和Nar能激活hPPARα,Dai、Nar和姜黄素(curcumin ,Cur)能激活hPPARγ1,而Dai、Ber、Nar、虎杖苷(polydatin, Pol)对hPPARδ有一定程度的激活作用。结论1)利用pMAL-p2x表达纯化体系,获得了大量、可溶的、电泳纯的MBP-hPPARsLBD和hPPARsLBD。经RBA鉴定,MBP的融合没有影响hPPARsLBD的配体结合功能;2)HPLC-UV法为一种新的,简便、经济、环保、有效的受体配体筛选方法,可用于研究受体配体结合反应;3)确认Ber、Nar是hPPARα的天然激动剂,Dai为hPPARα和hPPARγ1的双激动剂。此外,Cur对hPPARγ1, Dai、Ber、Nar、Pol对hPPARδ有一定程度的激活作用。提示Dai、Ber、Nar、Pol和Cur等中药单体至少通过激动hPPARs,调节下游多个靶基因表达,从而发挥调血脂、降血糖、抗肿瘤、抗炎等多种药理效应。
[Abstract]:Objective to establish a screening platform for human peroxisome prolifterator-activated receptors (hPPARs) ligand and screen the natural ligands of hPPARs from Chinese herbal monomers. Methods the total RNA was extracted from human liver cancer tissue, and the ligand binding domain (LBD) cDNA was amplified by RT-PCR. The cDNA was cloned into the downstream of the expression vector pMAL-p2x maltose binding protein (maltose binding hPPARs) coding gene sequence, and transformed into the binding. The host bacteria containing the recombinant plasmid were induced by optimal optimization, and the supernatant was broken by ultrasonic breakage. The product was purified by polysaccharide affinity resin, and purified MBP-hPPARsLBD was purified by Xa factor digestion, polysaccharide affinity resin chromatography and DEAE-52 anion exchange chromatography. The ligand binding function of MBP-hPPARsLBD and hPPARsLBD was identified by radioligand binding assays (RBA). The UV detection method of high performance liquid chromatography (high performance liquid chromatography and ultraviolet detection, HPLC-UV) and establish a screening system for hPPARsLBD ligands, and to try traditional Chinese medicine monomers were used, combined with the experimental radiolabeled ligand competition (radioligand binding competition assays classic, RBCA) method for HPLC-UV screening test results. The functional activity of the sifting drug was determined by the trans activation report gene method. Results the host strain containing recombinant plasmid was induced by optimum conditions (0.4 mmol/L, 30 h oscillating for 6 h), and about 20 mg MBP-hPPARsLBD was obtained from 1L culture, accounting for about 31% of total cytoplasmic protein. The purified MBP-hPPARsLBD and hPPARsLBD were obtained by purification and enzyme digestion. The yield was 14 mg/L and 5 mg/L, respectively, and the yield was 70% and 45%, respectively. RBA shows that the binding ligand function of MBP-hPPARsLBD and hPPARsLBD is equivalent. Based on the theory of molecular exclusion chromatography (Molecular Exclusion Chromatography, MEC), a HPLC-UV hPPARs ligand screening platform was established. This platform is used to treat reagents were screened, showed that berberine (berberine, Ber) and naringenin (naringenin, Nar) can specifically bind to hPPAR alpha, daidzein (daidzein, Dai) can specifically bind to hPPAR alpha and hPPAR gamma 1. The results of RCBA method are the same as that of HPLC-UV method. Trans activation of reporter gene method test of tested functional activity, traditional Chinese medicine monomer showed that Dai, Ber and Nar can activate hPPAR alpha, Dai, Nar and curcumin (curcumin, Cur) can activate hPPAR gamma 1, Dai, Ber, Nar, polydatin (polydatin, Pol) activity the degree of hPPAR. Conclusion 1) a large, soluble, electrophoretic pure MBP-hPPARsLBD and hPPARsLBD were obtained by using pMAL-p2x to express the purification system. Identified by RBA, MBP fusion hPPARsLBD did not affect the ligand binding function; 2) the HPLC-UV method is a new, simple and economical, environmental and effective receptor ligand screening method can be used to study the receptor ligand binding reaction; 3) confirm the Ber, Nar is hPPAR a natural agonist, Dai hPPAR alpha 1 double and hPPAR gamma agonist. In addition, the activation of hPPAR gamma 1, Dai, Ber, Nar, Pol on hPPAR delta is to a certain extent by Cur. It suggests that Dai, Ber, Nar, Pol and Cur can modulate the expression of multiple target genes at least through activating hPPARs, and play a variety of pharmacological effects such as lipid regulation, hypoglycemic, anti-tumor, anti-inflammatory and so on.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

【引证文献】