【作者】 徐莉春； 孙宏； 宋玲； 王心如；

【Author】 Xu Li-chun, Sun Hong, Song Ling, Wang Xinru (Institute of Toxicology of Jiangsu Province, School of Public Health, Nanjing Medical University, Nanjing 210029, China)

【机构】 南京医科大学应用毒理研究所；

【摘要】 [目的]建立以氯霉素乙酰化酶(CAT)为报告基因的雄激素受体(AR)报告基因试验。[方法]合成含雄激素效应元件(ARE)和启动子的小鼠乳腺癌病毒长末端重复序列(MMTV-LTR),定向插入报告基因质粒pCAT3-basic的Kpn Ⅰ和Bgl Ⅱ之间,构建受ARE调控的CAT报告基因质粒pMMTV-CAT,将表达AR的质粒AR/pcDNA3.1、重组质粒pMMTV-CAT和对照质粒pSV-β-Gal共转染CV-1,建立AR报告基因试验。以双氢睾酮(DHT)为阳性对照,检测该方法的筛选效率。[结果]10-8M的DHT明显诱导CAT的表达,诱导倍数达70。[结论]本研究建立的AR报告基因试验可作为化学物雄激素活性筛选的理想方法。更多还原

【Abstract】 [Objective] To develop an androgen receptor (AR) reporter gene assay in which the reporter gene is chloramphenicol acetyltransferase (CAT). [Methods] The oligonucleotides of the mouse mammary tumor viruslong terminal repeat (MMTV-LTR) containing androgen response element (ARE) and promoter were cloned into the kpn Ⅰ and Bgl Ⅱ sites of pCAT3-basic, creating pMMTV-CAT. The CV-1 was cotransfected with three plasmids including AR expression plasmid AR/pcDNA3.1 containing the cDNA of AR, reporter plasmid pMMTV-CAT containing the reporter gene regulated by an androgen response element (ARE) and the control plasmid pSV-β-Gal to create the AR reporter gene assay. The screening efficiency is determined by whether a reference androgen 5a-dihydrotestosterone (DHT) induced the CAT product. [Result] The CAT activity was induced obviously at 10-8M and the CAT activity is about 70 fold of that of the negative control. [Coclusion] The AR) reporter gene assay is useful in screening androgenic effect of chemicals.更多还原