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目的:克隆NKX3.1基因启动子并检测其启动子活性,为研究NKX3.1基因转录调控的基本机制奠定基础。方法:采用PCR方法从人基因组中扩增NKX3.1基因上游1.04 kb启动子片段并分别克隆到报道基因载体pGL3-basic和pEGFP-1中,通过转染细胞、荧光素酶活性测定及荧光显微镜下观察绿色荧光蛋白的表达,检测其启动子活性。结果:DNA测序结果证实克隆的1.04 kb启动子片段序列正确;pGL3-1.04 kb启动子转染LNCaP细胞后,双荧光素酶活性测定M1/M2=2.7,为pGL3-control活性的1.5倍,为pGL3-basic活性的50倍。表明克隆的人NKX3.1基因上游1.04 kb片段具有较强的启动子活性。为检测此1.04 kb启动子在不同组织细胞中的活性,分别将pGL3-1.04 kb启动子和pEGFP-1.04 kb启动子转染入不同的肿瘤细胞系,检测荧光素酶和绿色荧光蛋白的表达。结果显示1.04 kb启动子在前列腺癌细胞LNCaP中活性最高。采用TRANSFAC数据库分析,发现在1 040 bp片段内含有多种顺式作用元件,它们的功能性将需要进一步实验证明。结论:克隆的人NKX3.1基因上游1.04 kb片段具有较强的启动子活性,并且在前列腺癌细胞LNCaP中活性最高。
OBJECTIVE: To clone the promoter of NKX3.1 gene and test its promoter activity, which lays the foundation for studying the basic mechanism of transcription regulation of NKX3.1 gene. Methods: The 1.04 kb upstream of NKX3.1 gene was amplified from the human genome by PCR and cloned into the reporter gene vectors pGL3-basic and pEGFP-1, respectively. The transfected cells, luciferase activity assay and fluorescence microscopy The expression of green fluorescent protein (GFP) was observed and its promoter activity was measured. Results: DNA sequencing confirmed that the cloned 1.04 kb promoter fragment was correct. After transfecting LNCaP cells with pGL3-1.04 kb promoter, the dual luciferase activity assayed M1 / M2 = 2.7, which was 1.5 times of that of pGL3-control 50 times more pGL3-basic activity. The cloned human NKX3.1 gene upstream 1.04 kb fragment has a strong promoter activity. To detect the activity of this 1.04 kb promoter in different tissue cells, the pGL3-1.04 kb promoter and the pEGFP-1.04 kb promoter were transfected into different tumor cell lines to detect the expression of luciferase and green fluorescent protein. The results showed that the 1.04 kb promoter was the most active in prostate cancer cell line LNCaP. Using TRANSFAC database analysis, it was found that there are multiple cis-acting elements in the 1 040 bp fragment, and their functionality will require further experimental evidence. CONCLUSION: The 1.04 kb fragment upstream of the cloned human NKX3.1 gene has strong promoter activity and is most active in prostate cancer cell LNCaP.