三联组氨酸核苷酸结合蛋白2启动子报告基因质粒的构建和活性鉴定

发布时间：2018-07-25 06:15

【摘要】：目的三联组氨酸核苷酸结合蛋白2(Hint2,histidine triad nucleotide binding protein 2)在肝癌组织中低表达并与临床病理分期和病人预后密切相关,为研究Hint2的基因表达调控,构建人Hint2启动子荧光素酶报告基因质粒并鉴定活性。方法提取人基因组DNA,以其为模板,将含有Hint2启动子的不同长度的片段插入荧光素酶质粒p GL3-basic,后与β-半乳糖苷酶共转染He La细胞,转染后24h通过β-半乳糖苷酶实验检测转染效率,通过荧光素酶实验检测转录活性。结果质粒p GL3-h1(-6--463,457bp),p GL3-h2(-6--1255,1249bp)构建成功后,与β-半乳糖苷酶共转染人子宫颈癌He La细胞。转录活性=荧光素酶值/β-半乳糖苷酶值,结果显示p GL3-basic荧光素酶值/β-半乳糖苷酶值为147.3272,而p GL3-h1为67936,p GL3-h2为64234.19。空质粒与p GL3-h1,p GL3-h2之间差异具有统计学意义,而p GL3-h1,p GL3-h2之间差异不具有统计学意义。结论 Hint2位于转录起始位点上游-6至--463之间的启动子区域具有强转录活性。
[Abstract]:Objective to study the regulation of Hint2 gene expression in hepatocellular carcinoma (HCC) by low expression of Hint2 histidine triad nucleotide binding protein 2 (Hint2H histidine triad nucleotide binding protein 2), which is closely related to clinicopathological stage and prognosis. Construction of human Hint2 promoter luciferase reporter gene plasmid and identification of activity. Methods Human genomic DNA was extracted and inserted into luciferase plasmid pGL3-basic. then co-transfected with 尾 -galactosidase into He-La cells. The transfection efficiency was detected by 尾 -galactosidase assay 24 hours after transfection. Transcriptional activity was detected by luciferase assay. Results the plasmid p GL3-h1 (-6-463457bp) was successfully constructed and co-transfected with 尾 -galactosidase into human cervical cancer He-La cells. Transcriptional activity = luciferase / 尾 -galactosidase. The results showed that the value of p GL3-basic luciferase / 尾 -galactosidase was 147.3272 and p GL3-h1 was 67936 p GL3-h2 64234.19. There was significant difference between empty plasmids and p GL3-h1P GL3-h2, but there was no significant difference between p GL3-h1P GL3-h2. Conclusion Hint2 has strong transcriptional activity in the promoter region between 6-463 upstream of the transcription initiation site.
【作者单位】：福建省立医院药学部;福建省立医院肿瘤外科;
【基金】：福建卫生厅中青年骨干项目(No.2013-ZQN-JC-6) 福建科技厅面上项目(General program No.2015J01422) 福建省卫生厅青年项目(No.2014-1-4)
【分类号】：R735.7

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