CAJ | 학술논문

欧亚活血丹外源凝集素(Gleheda)是分离自欧亚活血丹(Glechoma hederacea)叶片中的一种糖基化植物新蛋白.如同其他糖基化蛋白,通过免疫学方法探测Gleheda的过程中通常受到一些不相干糖蛋白的妨碍,为此制定了抗Gleheda特异性多克隆抗体的纯化方案.免疫血清蛋白经硫酸铵选择性沉淀后,分别以Gleheda和刺槐外源凝集蛋白(RPA)结合在Sepharose 4B作为亲和配体,采用亲和层析法连续纯化2次,然后进一步采用离子交换层析Q Fast Flow提纯.经每一步骤提纯得到的抗体组分对Gleheda的特异性,均同时采用双向免疫扩散检验和Western blot分析.结果表明,以Gleheda为配体,亲和纯化制备得到的抗体组分对叶片粗提物中的许多植物(糖)蛋白仍然表现交叉反应.为除去由植物糖蛋白中的聚糖所引起这些非特异性交叉反应抗体,接着以RPA为配体再次进行亲和纯化,Western blot分析显示,抗体的特异性得到提高但并非除去了所有非特异性交叉反应的抗体.最后进一步采用离子交换层析制备得到仅抗Gleheda蛋白的特异性抗体组分,此抗体组分适用于免疫探测研究.该抗体纯化制备程序简易而高效,而且不需要昂贵的设备.
구아활혈단외원응집소(Gleheda)시분리자구아활혈단(Glechoma hederacea)협편중적일충당기화식물신단백.여동기타당기화단백,통과면역학방법탐측Gleheda적과정중통상수도일사불상간당단백적방애,위차제정료항Gleheda특이성다극륭항체적순화방안.면역혈청단백경류산안선택성침정후,분별이Gleheda화자괴외원응집단백(RPA)결합재Sepharose 4B작위친화배체,채용친화층석법련속순화2차,연후진일보채용리자교환층석Q Fast Flow제순.경매일보취제순득도적항체조분대Gleheda적특이성,균동시채용쌍향면역확산검험화Western blot분석.결과표명,이Gleheda위배체,친화순화제비득도적항체조분대협편조제물중적허다식물(당)단백잉연표현교차반응.위제거유식물당단백중적취당소인기저사비특이성교차반응항체,접착이RPA위배체재차진행친화순화,Western blot분석현시,항체적특이성득도제고단병비제거료소유비특이성교차반응적항체.최후진일보채용리자교환층석제비득도부항Gleheda단백적특이성항체조분,차항체조분괄용우면역탐측연구.해항체순화제비정서간역이고효,이차불수요앙귀적설비.
Glechoma hederocea agglutinin (Gleheda) is a novel glycosylated lectin isolated from the leaves of G. hederacea. Like other glycosylated proteins, the detection of Gleheda by immunological methods is often hampered by the cross-reactivity of the polyclonal antibodies with unrelated glycoproteins. Hence a protocol to purify monospecific polyclonal antibodies from a crude antiserum raised against Gleheda was developed. After selective ammonium sulfate precipitation and successive affinity chromatography on columns of Sepharose 4B with immobilized Gleheda and Robinia pseudoacacia agglutinin (RPA), respectively, ion-exchange chromatography on a column of Q Fast Flow was used for further purification. The specificity of the antibody fractions from each step was tested by double immunodiffusion assay and analyzed by Western blot. Results revealed that affinity chromatography of the immunoglobulin fraction on the immobilized Gleheda antigen yielded an antibody preparation that still cross-reacted with many proteins in leaf extracts. Depletion of nonspecific cross-reacting antibodies directed against the glycan part of the glycoprotein by affinity chromatography on immobilized RPA removed most but not all nonspecifically reacting antibodies. Only upon further purification by ion exchange chromatography an IgG fraction of monospecific antibodies that reacted exclusively with Gleheda could be obtained and accordingly was suitable for immunodetection studies. This antibody purification procedure promises simplicity and efficiency. In addition, this method does not require expensive facilities.