釉成熟蛋白多肽多克隆抗体的制备和应用

Preparation and application of polyclonal antibody against amelotin polypeptide

目的制备兔抗鼠釉成熟蛋白(amelotin)的多克隆抗体并进行鉴定和应用。方法对amelotin的蛋白质序列进行分析,选取一段氨基酸序列合成多肽并与钥孔戚血蓝素(KLH)偶联免疫新西兰大白兔,制备多肽抗体,ELISA检测抗体效价,Western blot法分析抗体特异性。免疫组织化学技术观察amelotin在小鼠下颌组织的表达情况。结果制备的兔抗amelotin抗体效价为1∶1 000 000,特异性高。免疫组织化学染色结果显示amelotin在3、7 d小鼠的磨牙釉质全层有强表达,并且在7 d小鼠下颌下腺的导管上皮细胞质中也有表达。结论成功制备了效价高、特异性好的兔抗amelotin抗体。

Objective To prepare and identify the rabbit anti-mouse polyclonal antibody against amelotin polypeptide.Methods The polypeptide was synthesized based on the bioinformatics analysis of amelotin,and coupled with keyhole limpet hemocyanin（KLH） for immunization. The amelotin polypeptide-KLH was injected into New Zealand rabbits to prepare the polyclonal antibody. ELISA technology was used to detect the titer of the antibody. The specificity of the polyclonal antibody was identified by Western blotting. The expression of amelotin in the submandibular tissue of mice was observed by immunohistochemistry. Results ELISA showed that the titer of amelotin antibody was 1∶1 000 000. Western blotting verified that the antibody had a high specificity. Immunohistochemistry indicated that amelotin was highly expressed in odontoprisis full-thick enamel of 3-day-old and 7-day-old mice,and also expressed in duct epithelial cytoplasm of submandibular glands of7-day-old mice. Conclusion The polyclonal antibody against amelotin polypeptide has been prepared successfully with high titer and high specificity.