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

Construction and Identification of luciferase Reporter Vectors Containing Human Hint2 Promoter

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。

OBJECTIVE Hint2 （histidine triad nucleotide binding protein2）, which is downregulated in hepatic cancer, have closely relate with clinicopathological stage and prognosis of patients. We construct and identify the activity of human hint2 promoter luciferase reporters to study regulation of gene expression of hint2. METHODS Extraction of genome DNA, with which as templates, We constructed pGL3-basic plasmids which contain different length of Hint2 promoter, and co-transfected it Hela with β-gal with lipofectamine 2000. Afetr transfection 24h, We detected the efficiency of transfection by β-gal and the activity of promoter＇ s different region by the luciferase values of pGL3- basic, pGL3-h1, pGL3-h2. RESULTS After these plasmids are constructed, cotransfected it Hela with β-gal, the transcriptional activity of pGL3-basic, pGL3-h1, pGL3-h2 is 147.33,67936. 00,64234. 19 respectively. There are statistically significant differences between transcriptional activity of pGL3-basic and pGL3-hl, pGL3-basic and pGL3-h2. The difference is not statistically significant between transcriptional activity of pGL3-h1 and pGL3- h2. CONCLUSION In summary,the highest transcriptive activity region of Hint2 promoter is from -6 to -463bp in front of transcriptional start site.