人多巴胺D2基因启动子区―350AG多态位点荧光素酶表达载体构建与鉴定及活性检测.docVIP

人多巴胺D2基因启动子区―350AG多态位点荧光素酶表达载体的构建与鉴定及活性检测 　　[摘要] 目的 探讨人多巴胺D2（DRD2）基因启动子区-350 bp位点单核苷酸多态性（rs1799978）对DRD2基因转录活性的影响，为进一步揭示DRD2基因启动子区单核苷酸多态性的功能及其对创伤应激障碍综合征易患性的分子机制奠定基础。 方法 以人全血基因组DNA为模板，分别扩增DRD2基因启动子区-350 bp处分别为A或G的目的片段（-1000 bp～+168 bp），与经过改建的报告基因空载体pmirGlo-promoter相连接，构建启动子区-350 bp处分别为A和G的重组荧光素酶报告基因表达载体，并通过限制性内切酶双酶切及测序进行鉴定，然后将构建好并经过鉴定的表达载体pmirGlo-promoter-A和pmirGlo-promoter-G分别与pRL-CMV瞬时共转染人胚肾癌细胞株HEK293，48 h后检测细胞中荧光素酶的相对表达量。 结果 双酶切及测序结果证实，成功构建了重组荧光素酶表达载体pmirGlo-promoter-A和pmirGlo-promoter-G；荧光素酶活性检测结果显示，pmirGlo-promoter-G的荧光素酶基因表达量显著高于pmirGlo-promoter-A，差异有高度统计学意义（P 0.01）。 结论 成功构建了pmirGlo-promoter-A/pmirGlo-promoter-G荧光素酶报告基因重组质粒，DRD2基因启动子区-350 bp为G时，基因转录活性较高，为A时，基因转录活性较低，说明rs1799978多态位点由A突变为G后，可能增强基因转录活性，该结果为进一步深入研究DRD2基因启动子区-350 bp单核苷酸多态性的生物学功能奠定了实验基础。 　　[关键词] 多巴胺受体D2；启动子区；单核苷酸多态性；荧光素酶活性 　　[中图分类号] R78 [文献标识码] A [文章编号] 1673-7210（2015）09（c）-0004-05 　　[Abstract] Objective To investigate the effects of single nucleotide polymorphism （SNP） in -350 bp （rs1799978） of Dopamine receptor D2 （DRD2） gene on transcription activity and analyze the mechanism of gene regulation. Methods The promoter region sequences of DRD2 gene A or G at site -350 bp （-1000 bp to +168 bp） were amplified using whole blood genomic DNA as template were inserted into the modified pmirGlo-promoter respectively. The recombinant vector and pmirGlo-Basic vector was co-transfected together with pRL-CMV vector containing renila luciferase reporter gene into primarily cultured HEK293. Firefly and Renila luciferase activities were analyzed 48 h later by use of Dual Luciferase reporter assay system. Results Restriction enzyme digestion and sequencing analysis confirmed the correct construction of recombinant expression vector pmirGlo-promoter-A/pmirGlo-promoter-G. The results of luciferase detection showed that the relative luciferase activity of pmirGlo-promoter-G was significantly higher than that of pmirGlo-promoter-A， the difference was statistically significant （P 0.01）. Conclusion Luciferase repo