论文部分内容阅读
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 construct the luciferase reporter gene vector pGL3-Basic by inserting the regulatory genes upstream of the adiponectin (AD) promoter of 1 100 bp in length (including the promoter, -1066 To + 4 bp) The luciferase reporter gene containing promoter regulatory sequence (pGL3-Basic-ADI1100) was used to study the regulation of adiponectin expression in Chinese hamster ovary cells (CHO). Methods: A 100 bp AD promoter fragment was amplified by PCR and ligated with PUC19T vector. The plasmid pUC19T-ADI1100 and plasmid pGL3-Basic of luciferase reporter gene were transfected into E. coli DH5a and amplified. And pGL3-Basic was digested with KpnI and XhoI respectively. The ADI1100 fragment and the large fragment of pGL3-Basic were digested by electrophoresis, and the fragment of ADI1100 was inserted into the luciferase Enzyme reporter gene pGL3-Basic, and transfected CHO cells, luciferase reporter gene activity. Results: The constructed plasmids contained upstream regulatory sequences of adiponectin promoter by digestion and gene sequencing. Transient transfection experiments showed that the transcriptional expression of AD promoter in CHO cells increased with time, after transfection, The dual reporter activity at 48 h was 30-fold more pGL3-Basic. CONCLUSION: The luciferase reporter gene was successfully constructed and provided the basis for the follow-up screening of traditional Chinese medicines that inhibit the effects of obesity.