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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 polyclonal antibodies against rabbit amelotin and identify and apply them. Methods The protein sequence of amelotin was analyzed. A polypeptide of amino acid sequence was synthesized and conjugated with keyhole limpet hemocyanin (KLH) to immunize New Zealand white rabbits to prepare peptide antibody. The antibody titer was detected by ELISA and the antibody specificity was analyzed by Western blot . Immunohistochemistry was used to observe the expression of amelotin in mouse mandibular tissue. Results The titer of rabbit anti-amelotin antibody was 1: 1 000 000 with high specificity. Immunohistochemical staining showed that amelotin was strongly expressed on the molars enamel layer on day 3 and day 7, and was also expressed on the ductal epithelial cytoplasm of the submandibular gland on day 7. Conclusion The rabbit anti-amelotin antibody with high titer and specificity was successfully prepared.