AD-004蛋白表达、抗体制备及其在睾丸和肾上腺组织中的表达分析  

Protein expression and preparation of polyclonal antibody of AD-004 and study on its expression in the adrenal and testis

作　　者：乔洁[1] 郑翠霞[2] 陈霞[1] 李雪松[1] 李圣贤[1] 马勤耘[1] 赵双霞[1] 袁国跃[1] 陈名道[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院内分泌代谢病科上海市内分泌代谢病研究所上海市内分泌代谢病临床中心 [2]上海交通大学医学院附属仁济医院急诊科

出　　处：《中华内分泌代谢杂志》2006年第4期367-371,共5页Chinese Journal of Endocrinology and Metabolism

基　　金：国家自然科学基金(30470816)

摘　　要：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 prepare rabbit antibody against mouse AD-004. by AD-004. expressed in the prokaryotic expression system and to identify its distribution in the testis and adrenal. Methods The full-length cDNA of mouse AD-004. was cloned into PET28 plasmid, and the protein was induced in E. coli BL21 bacteria by adding IPTG and then purified by Ni^2＋ -NTA column. The purified protein was used as an immunogen to prepare polyclonal antibody （pAb） of AD-004. The specificity of the antibody was detected by Western blotting. Immunohistochemical staining was performed in the mouse adrenal and testis via pAb of AD-004. Results Hisfused AD-004 was expressed efficiently in the prokaryotic system. Western blot analysis showed that the polyclonal antibody was duly bound to purified AD-004. with high specificity and sensitivity. AD-004. could be abundantly identified in the adrenal medulla and mainly expressed in the Leydig cells of testis. Conclusion The mouse protein of AD-004. is obtained from the prokaryotic expression system. The rabbit anti-AD-004, antibody has been prepared successfully. AD-004. protein is mainly localized in the interstitium of testis, suggesting that AD-004. may play a role in the synthesis of sex-steroid hormone.

关 键 词：AD-004蛋白 原核表达 腺苷酸激酶 抗体 多克隆 

分 类 号：R392[医药卫生—免疫学]