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目的以原核表达的小鼠肾上腺蛋白AD-004为免疫原,制备抗AD-004的兔多克隆抗体,并明确其在肾上腺、睾丸组织中的分布。方法将小鼠AD-004 eDNA克隆于原核表达载体PET28,经IPTG诱导AD-004的表达。用Ni2+-NTA纯化后的蛋白为免疫原制备抗AD-004多克隆抗体。通过Western印迹鉴定抗体特异性及效价,并用此抗体对小鼠睾丸、肾上腺组织进行免疫组织化学分析。结果此原核表达系统可高效表达AD-004蛋白,Western印迹结果显示所制备的抗体具有很高的效价和特异性,免疫组化提示AD-004蛋白在肾上腺的髓质表达较高,而在睾丸中主要表达于间质的Leydig细胞。结论本研究获得了小鼠AD-004原核表达蛋白,成功制备了AD-004多克隆抗体,并发现其在睾丸间质Leydig细胞表达最高,提示AD-004可能在睾丸性激素的合成过程中起重要作用。
Objective To construct the polyclonal antibody against AD-004 and to determine its distribution in the adrenal gland and testis by using prokaryotic expression product AD-004 as the immunogen. Methods The mouse AD-004 cDNA was cloned into the prokaryotic expression vector PET28 and the expression of AD-004 was induced by IPTG. Anti-AD-004 polyclonal antibody was prepared using Ni2 + -NTA purified protein as immunogen. Antibody specificity and titer were identified by Western blotting and the mouse testes and adrenal tissues were immunohistochemically analyzed with this antibody. Results The prokaryotic expression system could express AD-004 protein efficiently. Western blotting showed that the prepared antibody had high titer and specificity. Immunohistochemistry indicated that the expression of AD-004 protein in medulla of adrenal gland was high, Leydig cells, mainly expressed in interstitium, in testis. Conclusion The prokaryotic expression of AD-004 was obtained in this study. The polyclonal antibody of AD-004 was successfully prepared and found to be the highest expression in Leydig cells, suggesting that AD-004 may play an important role in the synthesis of testicular sex hormones effect.