【作者】 胡江丽；

【导师】 李家儒；

【作者基本信息】 武汉大学 ， 植物学， 2005， 硕士

【摘要】 本文以中国特有的甾体激素药源植物盾叶薯蓣为研究对象,通过薯蓣皂素免疫原的合成及其免疫效果分析,获得特异识别薯蓣皂素多克隆抗体,建立了薯蓣皂素斑点酶联免疫吸附检测方法(Dot-ELISA),并对盾叶薯蓣植株中薯蓣皂素进行了免疫组织化学定位与空间分布的研究。主要结果如下: 1.采用二环己基碳二亚胺(dicyclohexylcarbodiimide,DCC)法和水溶性碳二亚胺[1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,EDC]法,分别将琥珀酸酐化的薯蓣皂素与牛血清白蛋白(bovine serum albumin,BSA)偶联,得到不同的免疫原,通过免疫获得兔多克隆抗体,用间接竞争性Dot-ELISA方法分析比较这两种免疫原的免疫效果,结果表明,DCC法合成的薯蓣皂素免疫原免疫效果优于EDC法。 2.采用DCC法将琥珀酸酐化的薯蓣皂素与牛血清白蛋白偶联得到免疫原,通过免疫获得兔抗薯蓣皂素及其皂甙的多克隆抗体,建立了薯蓣皂素斑点酶联免疫吸附检测方法。本法对薯蓣皂素的检测范围为1μg/ml至250μg/ml,亦可通过检测盾叶薯蓣中总皂甙(检测范围0.1μg/ml到100μg/ml)得出薯蓣皂素的含量,可以简化薯蓣皂素含量的测定程序。与高效液相色谱法、比色法和重量法比较,薯蓣皂素的酶联免疫吸附检测方法具有样品制作简单、仪器设备要求低、灵敏度高的优点,可方便用于检测大批量样品的薯蓣皂素含量。 3.通过制备抗薯蓣皂素多克隆抗体,用免疫组织化学方法对盾叶薯蓣植株中的薯蓣皂素进行了定位。同时用EHSA方法对盾叶薯蓣植株叶、地上茎和根状茎中的薯蓣皂素进行了定量检测。结果:薯蓣皂素在盾叶薯蓣植株的叶,地上茎,限状茎都有分布。初步推测薯蓣皂素有可能是在叶肉细胞合成,然后通过胞间运输进入维管束的韧皮部,再由维管束的韧皮部进一步向下运输并在根状茎储藏。本文的结果为该植物的规范化种植与综合利用提供理论依据和技术支持。更多还原

【Abstract】 In this paper the Dioscorea zingiberensis C. H. Wright, a special Chinese species for steroid drugs manufacturing, was used as study material. An indirect competitive dot enzyme linker immunosorbent assay method of diosgenin was set up by multiclonal antibodies which was obtained through preparating of immunogens of diosgenin and their immuno-effects analysis; Immunohistochemical localization and interspace distribution of diosgenin in Dioscorea Zingiberensis C. H. Wright was investigated. The main results were as follows:l.The succinylatcd diosgenin was conjugated to bovine scrum albumin(BSA) by dicyclohexylcarbodiimide (DCC)-method and l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC)-mcthod separately. Rabbits were immunized with these two conjugates as immunogens to produce multiclonal antibodies, the multiclonal antibodies were evaluated by an indirect competitive Dot-ELISA. The results showed that the immuno-effect of immunogens made by DCC-method is better than that by EDC-method.2.Diosgenin-Bovine Serum Albumin conjugate was prepared by N,N’-dicyclohexylcarbodiimide method. After immunizing male New Zealand rabbits with this conjugate, we developed multi-antibodies against diosgenin and its glycosides. An indirect competitive dot enzyme linker immunosorbent assay(Dot-ELISA) method of diosgenin was set up. The detecting range for diosgenin is lμg/ml-250μg/ml, for glycoside, 0.1μg/ml-100μg/ml. By detecting glycosides to show the diosgenin content in Dioscorea zingiberensis C. H. Wright rhizome, we simplified the assay procedure. Compared with high-performance liquid chromatography, spectrometric and gravimetric assays, this assay is sensitive, simple and efficient for a large number of samples.3. The immunohistochemical localization of diosgenin in DioscoreaZingiberensis C. H. Wright was done after we developed multi-antibodies against diosgenin and its glycosides. The interspace distribution of diosgenin was examined by An indirect competitive enzyme linker immunosorbent assay (ELISA). We found that diosgenin was distributed in leaves, stem on the ground and rhizomorphous caudex under the ground. Immunohistochemical analysis revealed that diosgenin could be Synthesized in the cell of leaves, transported by phloem of caudex, and then storaged in rhizomorphous caudex under the ground at last. The results provided significant theoretical and technical basis for cultivation and synthetic exploitation of this kind of plant.更多还原