Study on a simple assay and purification system against active glycosides and scFV gene

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A new immunostaining method was found in this project. We named it eastern blotting. All compounds on TLC plate developed was transferred to the PVDF membrane and incubated in the NaIO_4 solution resulting in the cleavage of sugar moiety. When carrier protein was added, hapten-carrier protein conjugate was produced to be fixed on the membrane. Monoclonal antibody against the hapten was added and treated with secondary labeled antibody, then substrate. Double staining by the newly established eastern blotting using anti gisenoside Rb1 and Rg1 clearly indicated that the structures of ginsenosides may be easily suggested the type of aglycone, protopanaxatriol or protopanaxadiol, and moreover the number of sugar in a molecule comparing with the color stained and Rf value.Immunoaffinity column fixing monoclonal antibody makes it possible to isolate the hapten compound by one step. We succeeded the one-step isolation of ginsenoside Rb1 from the ginseng crude extract.ScFV gene was cloned from the total mRNA of hybrodoma secreting anti-solamargine monoclonal antibody. The gene was transformed into the plasmid of E. coli and expressed. The inclusion body which expressed scFV protein was refolded and purified by chelate affinity column. When compared the native IgG and scFV against solasodine glycosides, their closs-reactivities were completely same. We found a unique phenomenon which the concentration of solasodine glycosides increased by the induction of scFV gene into Solanum khasianum which produces solasodine glycosides. This methodology may be applied for the breeding of plant producing higher concentration of secondary metabolite without any constraction of biosynthetic enzyme genes.