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目的:表达并纯化小鼠釉成熟蛋白(Amelotin),制备多克隆抗体,并进行初步鉴定。方法:RT-PCR获得Amelo-tin成熟肽片段,构建pET32a-Amelotin,IPTG诱导表达Amelo-tin,纯化后免疫新西兰大白兔制备多克隆抗体,ELISA检测抗体滴度。Western blot和免疫组织化学鉴定抗体特异性。结果:成功构建了Amelotin重组表达载体pET32a-Amelotin,表达的重组蛋白纯化后免疫新西兰大白兔,得到的多克隆抗体ELISA显示抗体效价可以达到1∶12800。Western blot分析表明该抗体能特异结合Amelotin,免疫组化分析表明自制的抗体能特异的与Amelotin相互作用。结论:以纯化的Amelo-tin为免疫原,成功地制备了效价及特异性较高的兔抗Amelo-tin抗体,为进一步建立简便的Amelotin检测方法及研究Amelotin生物学功能奠定了良好的基础。
AIM: To express and purify mouse Amelotin and prepare polyclonal antibodies for preliminary identification. Methods: Amelo-tin mature peptide fragment was obtained by RT-PCR and pET32a-Amelotin was constructed. IPTG induced the expression of Amelo-tin, purified and immunized New Zealand white rabbits to prepare polyclonal antibody. ELISA titers were measured by ELISA. Antibody specificity was identified by Western blot and immunohistochemistry. Results: Amelotin recombinant plasmid pET32a-Amelotin was successfully constructed. The recombinant protein was purified and immunized New Zealand white rabbits. The obtained polyclonal antibody showed that the antibody titer reached 1:12800. Western blot analysis showed that the antibody can specifically bind to Amelotin, and immunohistochemistry analysis showed that the antibody can specifically interact with Amelotin. CONCLUSION: The rabbit anti-Amelo-tin antibody with high titer and specificity was successfully prepared with purified Amelo-tin as the immunogen, which laid a good foundation for further establishing a simple Amelotin assay and studying the biological function of Amelotin .