脂联素调控序列荧光素酶报告基因荧光素酶活性的分析

Cloning the Promoter of the Human Adiponectin Gene and Research on its Luciferase Activity

目的:通过将1 100 bp长度的人脂联素(adiponectin,AD)启动子上游的调控基因(包括启动子,-1066 To+4 bp)插入荧光素酶报告基因载体pGL3-Basic中,构建成含启动子调控序列的荧光素酶报告基因(pGL3-Basic-ADI1100),用于脂联素在中国仓鼠卵巢细胞(CHO)中的表达调控研究。方法:利用PCR技术扩增1 100 bp长度AD启动子片段,与PUC19T载体连接,将PUC19T-ADI1100质粒,及荧光素酶报告基因pGL3-Basic质粒转染大肠肝菌(DH5a)后扩增,提取并纯化PUC19T-ADI1100和pGL3-Basic;分别以KpnI,XhoI酶切pGL3-Basic;电泳并回收ADI1100片段和pGL3-Basic酶切大片段,在T4 DNA连接酶的作用下,将ADI1100片段插入荧光素酶报告基因pGL3-Basic中,并转染CHO细胞,检测荧光素酶报告基因活性。结果:通过酶切及基因测序的方法证实所构建质粒含有脂联素启动子上游调控序列;瞬时转染实验显示AD启动子在CHO细胞中的转录表达随时间的变化而升高,转染后48 h的双报告基因活性是pGL3-Basic的30倍。结论:该荧光素酶报告基因构建成功,为后续筛选有抑制肥胖作用的中药提供基础。

Objective： To clone the promoter of the adiponectin gene and investigate its transcriptional activity in the Chinese hamster ovary cells（CHO） cell lines. Method： The promoter of the human adiponectin gene was amplified from human genomic DNA by PCR, and then it was subcloned into PUC-19T and luciferase reporter gene.The Luciferase report systems with the promoter of the adiponectin gene were used to transfer CHO cells.The luciferase activities of the transferred cells were compared by luciferase assay. Result： The luciferase activity demonstrated that the constructed vector had the promotor activity.The CHO cells presented a stronger adiponectin promoter activity in 48 h than pGL-3basic vector. Conclusion： The human adiponecitin luciferase reporter gene vector has been constructed successfully, and it will become essential material for further study of obesity therapy with the traditional Chinese medicine.