摘要
目的构建含有人脂肪细胞型脂肪酸结合蛋白(FABP4)基因启动子荧光素酶报告基因质粒pGL3-Basic-FABP4-promoter,为脂类代谢相关药物研究提供基础。方法根据已知人的FABP4基因启动子序列设计引物,以人DNA为模板,用PCR法扩增出人FABP4基因启动子的DNA大片段。经过限制性内切酶KpnI和XhoI双酶切后插入到pGL3-Basic载体中;测序鉴定;然后将这些重组质粒瞬时转染到K293细胞中,并在24 h后检测其荧光素酶活性。结果构建的含荧光素酶基因的质粒,经酶切及测序鉴定结果显示FABP4启动子插入的位置和序列正确。转染细胞后检测其荧光显示:重组pGL3质粒转录活性较pGL3-Basic质粒明显增强(P<0.001)。结论成功构建了pGL3-Basic-FABP4-promoter质粒,为进一步研究FABP4基因的表达调控提供了工具和基础。
Objective To construct an FABP4 promoter recombined luciferase reporter gene vector, and to detect its function, thus providing a ba- sis for the research on lipid metabolism related medicines. Methods Primers were designed based on human FABP4 gene promoters, and FABPd promoters from human genome DNA was rephcated by PCR. Then FABP4 promoters were inserted into the pGL3-basic vector after double digestion by restriction enzyme KpnI and XhoI. Positive clones were identified by sequencing. The recombinant pGL3- basic-FABP4-promoter vector were transiently transfected into K293 cells. After 24 hours, the activity of luciferase was detected. Results A pGL3 luciferase reporter vector was con- structed. The result of sequencing and double digesting of recombined plasmid were completely correct. The results of luciferase detection after trans- fection showed that the transcriptional activity of recombinant pGL3 plasmid was obviously increased compared with that of pGL3- basic plasmid （P 〈 0.001）. Condusion A pGL3 luciferase reporter vector containing human FABP4 promoter gene was constructed successfully, which pro- vides a tool and basis for fttrther study ofFABP4 gene expression regulation.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2014年第2期166-169,共4页
Journal of China Medical University
基金
国家自然科学基金(81270568)
