摘要
目的:以鲁斯可皂苷元(ruscogenin,RUS)为配基制备亲和层析柱,并应用于免疫亲和纯化抗血清。方法:将鲁斯可皂苷元衍生为带有两个-COOH臂的丁二酸单酯衍生物(succinylatedruscogenin,RUS-2HS),与带有10碳原子链的琼脂糖凝胶EAHSepharose4B偶联,制成亲和层析柱;以制备的亲和层析柱纯化人工合成抗原RUS-2HS-BSA(牛血清白蛋白)免疫家兔产生的抗血清,得到特异性较高的抗体,并检测纯化抗体的纯度及特异性。结果:成功将RUS-2HS作为配体偶联至亲和介质上,偶联量为4.24mg.mL-1,偶联率为53.25%;竞争性抑制试验显示纯化抗体交叉反应率下降,特异性提高;SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示单一蛋白质条带,其相对分子质量约为51.2kD。结论:建立了以小分子天然活性产物为配基制备亲和柱的方法,应用于抗血清的纯化,获得的特异性抗体为探索该类难检测的活性成分的药理作用机制提供新的技术手段。
AIM： To prepare succinylated ruscogenin affinity chromatography column to be appliedfor the purification of Antiserum. METHODS： Suecinvlated ruscogenin was derived from ruscogenin and efficiently cross-linked to EAH-Sepharose 4B by an ann of 10-atom carbon chain as an affinity chromatography column. Antiserum was purified by the affinity chromatography using a gradient of NaCl buffer as an eluant. Moreover, the purified antibody was also determined with indirect Enzyme-linked immnunosorbent assay （EL1SA） and SDS-PAGE identification. RESULTS： RUN-2HS was successfully coupled to the matrices and the binding capacity of RUS--2HS was 4.24 mg·mL^-1 , the binding rate being 53.25%. SDS-PAGE identification of the purified antibody revealed a major protein band with a molecular weight of about 51.2 kD. CONCLUSION： A simple method of preparing of drug-mediated affinity chromatography column has been established in this study, which can be applied to the purification of antiserum. Furthermore, it can supply a new means in the active mechanism research of the active components.
出处
《中国天然药物》
SCIE
CAS
CSCD
2006年第5期363-367,共5页
基金
国家863计划"创新药物和中药现代化"重大专项2002AA2Z3209
江苏省自然科学基金BK99102
2005年江苏省高等学校研究生创新计划95号~~
关键词
鲁斯可皂苷元
亲和层析
抗体纯化
Ruseogenin
Affinity chromatography
Antiserum purification
