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

活性苷和scFV基因简易检测纯化系统的研究

• 批准号: 11470470
• 负责人: SHOYAMA Yukihiro
• 金额: $ 7.04万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470470/
• 关键词: monoclonal antibody; pharmacologically active saponin; eastern blotting; affinity column; one-step purification; scFV gene; transgenic plant; breeding; 活性配糖体; 高感度アツセイ系; ウエスタンブロッテイング; アフィニティークロマト; 小型化抗体遺伝子; 高感度アッセイ系; ウエスタンブロッティング; 発現

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.

本课题建立了一种新的免疫染色方法。我们称之为东方印迹法。将TLC板上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。当加入载体蛋白时，产生半抗原-载体蛋白缀合物以固定在膜上。加入抗半抗原的单克隆抗体，用第二标记抗体处理，然后用底物处理。用新建立的抗人参皂苷Rb 1和Rg 1的Eastern印迹法进行双重染色，结果表明人参皂苷的结构可以很容易地反映其苷元、原人参三醇或原人参二醇的类型，而且通过染色的颜色和Rf值可以很容易地反映出分子中糖的数目，用免疫亲和柱固定单克隆抗体可以一步分离出半抗原化合物。从人参粗提物中成功地一步分离到了抗茄边碱的抗茄边碱单克隆抗体（solamargine monoclonal antibody，Solamargine monoclonal antibody，Solamargine）基因，并从分泌抗茄边碱单克隆抗体的杂交瘤细胞总mRNA中克隆了ScFV基因。将该基因转化到E. coli中进行表达。表达scFV蛋白的包涵体经复性、螯合亲和柱纯化。当比较天然IgG和scFV对澳洲茄定糖苷时，它们的紧密反应性完全相同。我们发现了一个独特的现象，即通过将scFV基因诱导到产生澳洲茄苷的茄属植物中，澳洲茄苷的浓度增加。该方法可用于选育不受生物合成酶基因干扰的高浓度次生代谢产物产生植物。

项目成果

O.Morinaga et al.: "Production of monoclonal antibody against a major purgative componet. sennoside B, their charactrization and ELISA"Analyst. 125. 1109-1113 (2000)

O.Morinaga 等人：“针对主要泻药成分番泻苷 B 的单克隆抗体的生产、其表征和 ELISA”分析员。

N.Fukuda et al.: "Isolation of Pharmacologically active saponin ginsenoside Rb, from ginseng by immuno affinity column chromatography"J.Nat.Prod.. 63(2). 283-285 (2000)

N.Fukuda 等人：“通过免疫亲和柱色谱从人参中分离药理活性皂苷人参皂苷 Rb”J.Nat.Prod.. 63(2)。

W. Putakun et al: "Rapid separation of solasodine glycoside by an immunoaffinity colun using anti-solamorgine monoclonal antibody"Cytotechnology. 31. 151-156 (1999)

W. Putakun 等人：“使用抗索拉莫碱单克隆抗体通过免疫亲和柱快速分离索拉索定糖苷”细胞技术。

N.Fukuda et al.: "Isolation of pharmacologically active saponin geinsenoside Rb1 from ginseng by immunoaffinity column chromatography"J. Nat. Prod.. 63(2). 283-285 (2000)

N.Fukuda 等：“通过免疫亲和柱色谱法从人参中分离具有药理活性的皂苷 geinsenoside Rb1”J.

Y.Shoyama, H.Tanaka, N.Fukuda, S.J.Shan, K.Muraoka: "ELISA, immunoaffinity column, western blotting and immunocytolocalization using MAbs"Res.Adv. in Phytochemistry. 1. 83-104 (2000)

Y.Shoyama、H.Tanaka、N.Fukuda、S.J.Shan、K.Muraoka：“使用 MAb 进行 ELISA、免疫亲和柱、蛋白质印迹和免疫细胞定位”Res.Adv。