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目的:构建小鼠谷胱甘肽S转移酶K1(mGSTK1)启动子荧光素酶报告基因质粒,并分析其活性。方法:根据GeneBank中mGSTK1基因序列设计引物,应用PCR技术扩增mGSTK1启动子片段,插入pGL3-basic载体,获得mGSTK1启动子报告基因质粒pGL3-mGSTK1-2046。再以pGL3-mGSTK1-2046为模板,进一步构建了2个5’端部分缺失的报告基因质粒:pGL3-mGSTK1-936和pGL3-mGSTK1-76。将构建的报告基因质粒瞬时转染不同的细胞株3T3-L1和人胚肾(HEK)293,48 h后检测荧光素酶的活性。结果:所构建的质粒经酶切、测序鉴定正确。pGL3-mGSTK1-2046在3T3-L1和HEK293中均有不同程度的转录活性。将pGL3-mGSTK1-2046、pGL3-mGSTK1-936和pGL3-mGSTK1-76转染HEK293细胞,结果显示pGL3-mGSTK1-936转录活性最高,而pGL3-mGSTK1-76转录活性显著降低。结论:成功构建mGSTK1启动子荧光素酶报告基因质粒;翻译起始点上游的-971～-111 bp片段是mGSTK1启动子的重要区域。为深入了解mGSTK1启动子的调控机制以及今后对代谢性疾病的治疗提供一定的实验基础。
OBJECTIVE: To construct a mouse luciferase reporter plasmid containing glutathione S-transferase K1 (mGSTK1) promoter and analyze its activity. Methods: According to the sequence of mGSTK1 gene in GeneBank, primers were designed. The mGSTK1 promoter fragment was amplified by PCR and inserted into the pGL3-basic vector to obtain the mGSTK1 promoter reporter plasmid pGL3-mGSTK1-2046. Two pGL3-mGSTK1-936 and pGL3-mGSTK1-76 partial deletion reporter plasmids were further constructed by using pGL3-mGSTK1-2046 as a template. The constructed reporter plasmids were transiently transfected into different cell lines 3T3-L1 and human embryonic kidney (HEK) 293 for 48 h, then luciferase activity was detected. Results: The constructed plasmid was digested and sequenced. pGL3-mGSTK1-2046 in 3T3-L1 and HEK293 have varying degrees of transcriptional activity. Transfection of pGL3-mGSTK1-2046, pGL3-mGSTK1-936 and pGL3-mGSTK1-76 into HEK293 cells showed that the transcriptional activity of pGL3-mGSTK1-936 was the highest and the transcriptional activity of pGL3-mGSTK1-76 was significantly reduced. CONCLUSION: The mGSTK1 promoter luciferase reporter gene plasmid was successfully constructed. The -971 ~ -111 bp fragment upstream of the translation initiation site is an important region of the mGSTK1 promoter. In order to further understand the regulation mechanism of mGSTK1 promoter and provide some experimental basis for the future treatment of metabolic diseases.