CYP21和CYP21P基因启动子序列对绿色荧光蛋白基因表达的影响  

Expression of Green Fluorescent Protein Vector by Promoter sequence of CYP21 Gene and CYP21P Gene

作　　者：韩蓓[1] 王秀敏[1] 张雅芬[2] 顾学范[1] 

机构地区：[1]上海第二医科大学附属新华医院,上海200092 [2]上海市儿科医学研究所,上海200092

出　　处：《Acta Genetica Sinica》2002年第5期396-401,T002,共7页

基　　金：国家自然科学基金项目 (No :3 9870 778);上海市卫生系统百人计划项目资助 (No :98BR0 3 5 )~~

摘　　要：特异性扩增CYP2 1基因和CYP2 1P基因启动子区域 770bp～ 1bp片段 ,去除pEGFP N1载体中的CMV启动子 ,构建含CYP2 1基因启动子的pEGFP N1载体 (pCYP2 1)和CYP2 1P基因启动子的pEGFP N1载体 (pCYP2 1P) ,分别将上述两种构建载体、野生型pEGFP N1(阳性对照 )质粒及阴性对照转染入肾上腺皮质来源的Y1细胞系中 ,用倒置荧光显微镜 ,以及激光共聚焦显微镜等方法观测绿色荧光蛋白的表达。转染后 ,在荧光倒置显微镜下首次发现Y1细胞中出现绿色荧光蛋白的时间阳性对照为 3小时 ,pCYP2 1为 7小时 ,pCYP2 1P与阴性对照 (未转染任何载体的Y1细胞 )始终未观测到绿色荧光蛋白。激光共聚焦显微镜显示 ,阳性对照绿色荧光蛋白表达强于pCYP2 1,pCYP2 1P与阴性对照始终未观测到绿色荧光蛋白。阳性对照和pCYP2 1的绿色荧光蛋白在胞核中的荧光强度高于胞浆。上述结果进一步表明 ,含有CYP2 1和CYP2After amplification of PCR fragment of -770bp～-1bp in promoters of CYP21 gene and CYP21P gene respectively, the CMV promoter was cut off from pEGFP N1, the vectors were constructed, in which contained promoter areas in CYP21 gene ( pCYP21 ) and CYP21P gene ( pCYP21P ). All pCYP21? pCYP21P ? pEGFP N1(positive control) and negative control were transfected respectively into steroidogenic Y1 cell line, and were observed by inverted fluorescent microscopy and laser confocal microscopy. After transient transfection, the cells placed on inverted fluorescent microscopy. The appearance of GFP expression observed is as follows: pEGFP N1 at 3 hours; pCYP21 at 7 hours. However, neither pCYP21P nor negative control expressed GFP. Laser confocal microscopy showed that pEGFP N1 and pCYP21 produced GFP. pEGFP N1 is stronger than pCYP21 , but none of pCYP21P and negative control expressed GFP. Different distribution of GFP in Y1 cell could be seen of pEGFP N1 and pCYP21 , and the intensity of GFP in nucleus is stronger than cytoplasm. Our results further confirm that there is a significantly difference of GFP expression in Y1 cell line by promoters of CYP21 and CYP21P .

关 键 词：基因表达 CYP21基因 YP21P基因 启动子 绿色荧光蛋白载体 Y1细胞系 肾上腺皮质增生 CAH 

分 类 号：Q786[生物学—分子生物学]