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

本项目发现了一种新的免疫染色方法。我们将其命名为东方印迹。TLC板上显像的所有化合物转移到PVDF膜上，并在NaIO_4溶液中孵育，导致糖部分裂解。加入载体蛋白后，产生半抗原-载体蛋白偶联物固定在膜上。加入抗半抗原的单克隆抗体，用二标记抗体处理，然后用底物处理。采用新建立的抗人参皂苷Rb1和Rg1双染色方法，通过染色颜色和Rf值的比较，可以很容易地判断人参皂苷的糖元类型、原人参皂苷醇或原人参皂苷二醇的结构，以及分子中糖的数量。免疫亲和柱固定单克隆抗体使得一步分离半抗原化合物成为可能。从人参粗提物中一步分离得到了人参皂苷Rb1。从分泌抗太阳碱单克隆抗体的淋巴瘤总mRNA中克隆出ScFV基因。将该基因转入大肠杆菌质粒表达。用螯合亲和柱对表达scFV蛋白的包涵体进行再折叠和纯化。将天然IgG和scFV与茄地定苷进行比较，它们的亲和反应性完全相同。结果表明，将scFV基因导入产生茄碱苷的哈苏茄中，茄碱苷的浓度明显升高。该方法可用于不需要构建生物合成酶基因而产生较高浓度次生代谢物的植物育种。

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.