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[1] 杨爱玲[1] 章锦才广东省口腔医院口腔内科 殷春一[1] 张蕴惠[1] 张静仪[2] 

机构地区：[1]四川大学华西口腔医院牙周科,610041 [2]四川大学华西口腔医学院重点实验室

出　　处：《华西口腔医学杂志》2003年第2期130-132,共3页West China Journal of Stomatology

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_2 0 0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_5 2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_2 0 0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 0 0 0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。Objective\ To prepare the polyclonal antibody to amelogenin.Methods\ The fetal porcine dental enamel was 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 adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific 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 at molecular weights of 15 kD and 13 kD apparently, 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.\;

关 键 词：猪 发育期 牙胚釉原蛋白 分离 纯化 多克隆抗体 抗釉原蛋白多克隆抗体 DE-52纤维素 

分 类 号：R781.4[医药卫生—口腔医学]