猪发育期牙胚釉原蛋白的分离纯化及其多克隆抗体的制备
Extraction and Purification of Porcine Amelogenin and Preparation for the Polyclonal Amelogenin Antibody
作者：徐琛蓉,杨爱玲,章锦才广东省口腔医院口腔内科,殷春一,张蕴惠,张静仪
Author：XUChenrong*,YANGAiling,ZHANG Jincai,et al.
收稿日期：2003-04-25 年卷（期）页码：2003,21(02):130-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：釉原蛋白,猪,抗釉原蛋白多克隆抗体,SephadexG-200,DE-52纤维素,
Key words：amelogenin,porcine,anti-amelogenin antibody,Sephadex G-200,DE-52 cellulose,
基金项目：
中文摘要
目的 制备猪釉质蛋白多克隆抗体,为釉原蛋白的检测提供条件。方法 选择1月龄乳猪,分离埋于上下颌骨内的牙胚,刮取牙胚表面干酪样尚未完全矿化的釉质基质,通过盐酸胍抽提及SephadexG-200柱层析分离纯化猪发育期牙胚釉原蛋白,联合免疫家兔,抗血清经DE-52纤维素纯化,并经ELISA测定抗体效价。结果 SDS-PAGE电泳结果发现采用SephadexG-200葡聚糖凝胶过滤能达到较为理想的釉原蛋白分离纯化效果,用所提纯的猪釉原蛋白免疫家兔,成功地制得兔抗猪釉原蛋白抗血清,抗血清稀释达1∶32 000时,采用ELISA法仍有明显的抗原抗体反应。结论 本实验成功制备得到抗釉原蛋白多克隆抗体,为釉原蛋白的检测提供了条件。
英文摘要
Objective To prepare the polyclonal antibody to amelogenin.Methods The fetal porcine dental enamelwas collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7·4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund′s adjuvantwas then used to immunize the rabbits subcutaneously or intravenously. The spe- cific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test.Results The Gel filtration showed that amelogenin components is atmolecularweights of 15 kD and 13 kD apparent- ly, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1∶ 1000.Conclusion The antibody prepared in this study can be used for the detection of amelogenin.
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