转录因子Ets-1表达载体的构建及调控小鼠成釉细胞MMP-20的表达  

Construction of Expression Vector of Transcription Factor Ets-2 and Regulating the Expression of Matrix MetaUoproteinase-20

作　　者：孙霞[1] 付显俊[2] 李江[1] 高玉光[3] 

机构地区：[1]潍坊医学院口腔医学研究所,山东潍坊261053 [2]潍坊市益都中心医院口腔科 [3]滨州医学院口腔医学院

出　　处：《潍坊医学院学报》2012年第3期186-188,I0003,共4页Acta Academiae Medicinae Weifang

基　　金：国家自然科学基金资助课题（课题编号：30973327）,山东省自然科学基金资助课题（课题编号：ZR2010HM076）

摘　　要：目的构建转录因子Ets-1的pcDNA331／myc-HisA真核表达载体，转染小鼠成釉细胞，检测Ets-1调控小鼠成釉细胞MMP-20的表达，为研究Ets-1在牙釉质发育过程中的重要作用奠定基础。方法以从小鼠成釉细胞中提取的总RNA逆转录所得eDNA为模板，用RT-PcR法扩增得出含有kpnI和BamHI的Ets-1目的基因连接到pcDNA3．1／myc-HisA真核表达载体上。将重组质粒瞬时转染到小鼠成釉细胞中观察，并通过实时定量RT-PCR的方法检测MMP-20mRNA的相对表达量。结果①经过PCR引物扩增得到-约1399bp的基因片段，重组质粒pcDNA3．1／myc．HisA-Ets-l双酶切进行初步鉴定后，与GenBank登录基因对比显示生物公司测序结果完全正确。（爹重组质粒pcDNA3．1／myc-HisA-Ets-1转染小鼠成釉细胞后（以转染空载体pcDNA3．1／myc-HisA作为对照），实时定量RT-PCR检测MMP-20tuRNA的相对表达量上升明显。结论成功构建了Ets-1的真核表达载体，初步研究证明小鼠成釉细胞核中Ets-1可以上调MMP-20基因表达水平。Objective To construct eukaryotic Ets-1 ,on conduction path of mice ameloblasts to test Ets-1 expression vector cDNA3, 1/myc-HisA of transcription factor regulates MMP-20 gene expression in ameloblasts,and lay a foundation to study the significance of Ets-1 in developing dental enamel. Methods The eDNA coding sequence of Ets- 1 containing the digestion site of kpn I and BamH I was amplified by RT-PCR from ameloblasts and then was inserted into pcDNA3.1/myc-HisA vector. The recombinant vector was transfected into ameloblasts instantaneously,then checked on the level of the comparative expression of MMP-20 mRNA with real time RT-PCR. Results ①The amplified gene fragment was 1399bp. Restriction enzyme digestion and sequencing showed that pcDNA3, 1/mye-HisA-Ets-I was con- strutted correctly. And it was confirmed by sequencing which was consistent with samples in GenBank. ②By real time PCR,compared to the control group（pcDNA3. 1/myc-HisA）. The express of MMP-20 rised obviously after trasfected pcDNA3, 1/myc-HisA-Ets-1plasmid into ALC. Conclusion The eukaryotic expression vector Ets-1 is constructed suc- cessfully. Preliminary studies has shown that Ets-I can up-regulate MMP-20 gene expression in ameloblasts.

关 键 词：转录因子ETS-1 基质金属蛋白酶-20 RT-PCR 实时定量RT-PCR 转染 

分 类 号：R781[医药卫生—口腔医学]