采用ODS、MCI gel CHP20以及硅胶等柱色谱技术并结合半制备液相对茯苓95%乙醇提物进行分离纯化,运用NMR、MS、IR、计算NMR等方法,对分离得到的化合物进行结构鉴定。从茯苓中共分离得到7个化合物,分别鉴定为20S-2β,3α,15α,19,20-羟基...采用ODS、MCI gel CHP20以及硅胶等柱色谱技术并结合半制备液相对茯苓95%乙醇提物进行分离纯化,运用NMR、MS、IR、计算NMR等方法,对分离得到的化合物进行结构鉴定。从茯苓中共分离得到7个化合物,分别鉴定为20S-2β,3α,15α,19,20-羟基-孕甾-7-烯(1)、去氢齿孔酸乙酰酯(2)、齿孔酸乙酰酯(3)、去氢齿孔酸(4)、齿孔酸(5)、去氢茯苓酸(6)、茯苓酸(7)。化合物1为新孕甾烷类化合物。展开更多
Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism a...Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body’s homeostatic mechanisms, we for the first time, report successful prokary- otic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.展开更多
文摘采用ODS、MCI gel CHP20以及硅胶等柱色谱技术并结合半制备液相对茯苓95%乙醇提物进行分离纯化,运用NMR、MS、IR、计算NMR等方法,对分离得到的化合物进行结构鉴定。从茯苓中共分离得到7个化合物,分别鉴定为20S-2β,3α,15α,19,20-羟基-孕甾-7-烯(1)、去氢齿孔酸乙酰酯(2)、齿孔酸乙酰酯(3)、去氢齿孔酸(4)、齿孔酸(5)、去氢茯苓酸(6)、茯苓酸(7)。化合物1为新孕甾烷类化合物。
文摘Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body’s homeostatic mechanisms, we for the first time, report successful prokary- otic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.
