激活转录因子-2调控小鼠成釉细胞基质金属蛋白酶-20分子机制的初步研究

    Preliminary study of the molecular mechanisms of activating transcription factor-2 regulating matrix metalloproteinase-20 in mice ameloblasts

    • 摘要: 目的:通过克隆激活转录因子-2(ATF-2)基因以及真核表达载体的构建,初步研究ATF-2对小鼠成釉细胞基质金属蛋白酶-20(MMP-20)基因表达的影响。方法:根据引物设计原则设计ATF-2基因逆转录-聚合酶链反应(PCR)引物,从小鼠成釉细胞中提取总RNA逆转录所得cDNA为模板,进行PCR法扩增。得出含有Hind Ⅲ 和xhol酶切位点的ATF-2目的基因连接到pcDNA 3.1/myc-HisA真核表达载体上;用重组质粒转染小鼠MMP-20基因,观察其对MMP-20基因转录活性的影响。结果:经过PCR引物扩增得到1 463 bp基因片段,将获得的重组质粒pcDNA 3.1/myc-HisA-ATF-2双酶切分析鉴定,测序结果与Gen Bank登录基因序列完全一致;双荧光素酶结果显示ATF-2可以促进MMP-20启动子的表达(P<0.01),且在-825~+23(848 bp)启动子区段促进作用最明显。结论:成功实现了ATF-2基因克隆及真核表达载体的构建,并发现ATF-2对MMP-20启动子区域活性表达起正向调节作用。

       

      Abstract: Objective: To clone the activating transcription factor-2(ATF-2) gene,then construct the recombinant eukaryotic expression vector with ATF-2,and explore the effects of ATF-2 on the gene expression of matrix metalloproteinase-20(MMP-20) in mice ameloblasts.Methods: The reverse transcription PCR primers of ATF-2 were designed.The total RNA from the mice ameloblasts was extracted to obtain cDNA for PCR amplification.The ATF-2 gene containing Hind Ⅲ and xhol restriction sites was inserted into the eukaryotic expression vector(pcDNA 3.1/myc-HisA).The effects of ATF-2 on the transcriptional activity of MMP-20 was observed after transfecting the recombinant plasmid.Results: One thousand four hundred and sixty-three bp gene fragment was amplified through PCR.The recombinant eukaryotic expression vector with ATF-2(pcDNA 3.1/myc-HisA-ATF-2) was successful constructed by the verification of dual enzyme digestion and sequencing.The luciferase test showed that ATF-2 gene could promote the expression of MMP-20 promoter(P<0.01).Conclusions: The recombinant pcDNA 3.1/myc-HisA-ATF-2 expression vector is successful constructed.ATF-2 can positive regulate the promoter activity of MMP-20.

       

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