Pharmacognosical study on secondary metabolites

Clonal micropropagation on various medicinal plants was set up resulting in the regenerated plants which possessed a homogeneous quality. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determine d by matrix-assisted laser desorption/ionization of mass spectrometry. A hp bridoma secreting monoclonal antibody (MAb) was produced by fusing splenocy tes immunized with an antigene-BSA conjugate with mouse myeloma cells. Comp etitive enzyme-linked immunosorbent assay (ELISA) using MAb was set up as a high sensitive, specific and reproducible qualitative method. A method of determination for ginsenosides by using a unique western blotting was estab lished. Immunoaffinity column chromatography using an anti-ginsenoside Rb1M Ab has made possible a single-step separation of ginsenoside Rbl from a cru de ginseng extract. Single chain Fv gene of anti-forskolin MAb was prepared from mRNA of hybridoma secreting anti-forskolin MAb and cloned. Gene was c onstructed into a pET-28a(+) vector producing a scFv protein. Modeling of f orskolin and scFV was investigated. THCA synthase was purified from the hom ogenate of Cannabis sativa leaves on successive column chromatographies. TH CA synthase was confirmed to be homogeneity having 75 kDa. To obtain the co rresponding cDNA clone of THCA synthase, a set of degenerate promers was co nstructed based on N-terinal and internal amino acide sequences of THCA syn thase. The 5' and 3' ends of cDNA were amplified by RACE. A full sequencing has been determined to be corded a polypeptide having 545 amino acid resid ues. The cDNA clone was expressed in yeast system via PUC19 vector resultin g in THCA synthase activity.