牛CART基因启动子片段重组载体构建

Construction of recombinant vector of bovine CART gene promoter fragment

中文关键词: CART  启动子  双荧光素酶报告基因

英文关键词: CART  the promoter  dual luciferase reporter gene

中文摘要:

      目的 构建以及鉴定可卡因—苯丙胺调节转录肽（cocaine-and amphetamine regulated transcript peptides, CART）基因启动子片段双荧光素酶报告载体，为后续筛选CART基因核心启动子奠定试验基础。方法 美国国家生物技术信息中心(national center for biotechnology information, NCBI)基因数据库（登录号：NC_037347.1）获取牛CART基因序列，经聚合酶链式反应（PCR）扩增启动子片段，经双酶切后连接pGL3-Basic载体。将pGL3-1222载体、pGL3-Control载体和pRL-TK载体转染293T细胞，检测细胞相对荧光活性。结果 pGL3-1222载体测序结果显示与目标序列一致，证明载体构建成功；转染293T细胞后，细胞状态良好且绿色荧光分布均匀、强度适中，则转染操作无误；检测相对荧光活性显示pGL3-1222组相对荧光活性显著高于pGL3-Basic，则证明CART基因启动子片段具有活性。结论 CART基因启动子-1200 bp — +22 bp区域双荧光素酶载体构建成功。

英文摘要:

      Objective To construct and identify a double luciferase reporter vector for promoters of cocaine-and amphetamine regulated transcript peptides (CART) gene. It lays the experimental foundation for screening the core promoter of CART gene. Methods national center for biotechnology information (NCBI) Gene Database (Accession No.: NC_037347.1) obtained the bovine CART gene sequence, amplified the promoter fragment by polymerase chain reaction (PCR), and ligated the pGL3-Basic vector after double digestion. 293T cells were transfected with pGL3-1222 vector, pGL3-Control vector and pRL-TK vector, and the relative fluorescence activity of the cells was detected. Results The sequencing results of pGL3-1222 vector were consistent with the target sequence, indicating that the vector was successfully constructed. After transfection, 293T cells were in good condition with uniform green fluorescence distribution and moderate intensity, indicating that the transfection operation was correct. The relative fluorescence activity of pGL3-1222 group was significantly higher than that of pGL3-Basic, which proved that the CART gene promoter fragment was active. Conclusion The dual luciferase vector in the -1200 bp -- +22 bp region of CART gene promoter was successfully constructed. This experiment will create experimental conditions for screening the core promoter of CART gene in the future.