摘要
【目的】合成放射诱导启动子序列并构建其调控双自杀基因的真核表达质粒pcDNA3.1(+)/E-CDglyTK。【方法】利用人工寡核苷酸片段合成放射诱导启动子,以绿色荧光蛋白(GFP)作为报告基因转染Tca8113细胞,经流式细胞仪鉴定其辐射诱导特性。将pCEA-CDglyTK中双自杀基因CDglyTK亚克隆到pcDNA3.1(+),然后把合成启动子插入到CDglyTK上游构建质粒pcDNA3.1(+)/E-CDglyTK并酶切鉴定,阳离子脂质体介导其转染舌鳞癌Tca8113细胞,RT-PCR观察CDglyTK表达及诱导放疗的增效作用。【结果】合成启动子序列分析与设计序列完全一致;低剂量放射线照射可诱导这种人工启动子增强GFP在Tca8113细胞中的表达;重组质粒的酶切图谱与预期一致;3Gy放疗显著增强CDglyTK在Tca8113细胞中的表达。【结论】成功合成放射诱导启动子并构建由其调控双自杀基因的真核表达质粒pcDNA3.1(+)/E-CDglyTK,为进一步研究肿瘤放射-基因治疗奠定了基础。
[Objective] To synthesize the radiation-inducible promoter and construct eukaryotie cell expression recombinant pcDNA3.1（＋）/E-CDglyTK. [Methods] The radiation-inducible promoters were synthesized with manmade complementary oligonucleotides. The enhancer regions of the CMV IE gene promoter in pCGFP were replaced by the synthetic promoters to construct the reporter plasmids pE-GFP. Green fluorescence protein （GFP） gene was used as the reporter gene to analyze the radiation-inducible property. CDglyTK gene in pCEA-CDglyTK was subcloned into pcDNA3.1 （＋）, then the synthetic promoter was inserted before the CDglyTK gene. CDglyTK gene expression in Tca8113 was detected by RT-PCR. [Results] DNA sequencing analysis proved that the sequence of the radiation-inducible promoter was totally consistent with the designing sequence. The GFP expression in Tea8113 cells transfected with pE-GFP was increased by induction of low dose γ-radiation. The digested product of recombinant peDNA3.1 （＋）/E-CDglyTK proved that the synthetic promoter and CDglyTK gene were subeloned into pcDNA3.1（＋） exactly. 3 Gy induction therapy markedly increased CDglyTK gene expression. [Conclusion] The synthetic promoters are responsive to low dose of ionizing radiation and the CArG elements can serve as a molecular switch to enhance down-stream gene expression. The peDNA3.1 （＋）/E-CDglyTK eukaryotie cell expression recombinant was successfully constructed.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2005年第5期502-505,共4页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金资助项目(30271423)
广东省自然科学基金资助项目(021865)
