基因突变对RUNX2蛋白亚细胞定位的影响  

Effect of the RUNX2 Gene Mutations on Subnuclear Localization of the RUNX2 Protein Mutants

作　　者：梁国健 谢宝仪[2,3] 轩东英[2,3] 

机构地区：[1]阳江市人民医院口腔科,广东阳江529500 [2]广东省口腔医院 [3]南方医科大学附属口腔医院

出　　处：《广东牙病防治》2011年第2期68-71,共4页Journal of Dental Prevention and Treatment

基　　金：广东省自然科学基金博士启动课题资助(9451026003003833);高等学校博士学科点专项科研基金(20100101120114)

摘　　要：目的拟建立包含基因突变位点的真核表达载体,通过细胞转染了解RUNX2突变体亚细胞定位的改变。方法从患者全血中提取总RNA,通过反转录酶-聚合酶链反应获得目的片段,定向克隆获得包含基因突变位点及野生型位点的pEGFP-C1-RUNX2真核表达载体,通过瞬时转染NIH3T3成纤维细胞,显微镜下观察突变蛋白的亚细胞定位变化。结果成功构建包含突变位点及野生位点的真核表达载体pEGFP-C1-RUNX2,转染细胞24 h后,在荧光显微镜下观察转染细胞内的荧光蛋白表达情况,转染野生型RUNX2基因的细胞仅在胞核内表达绿色荧光,胞浆内无绿色荧光表达;分别引入突变位点c.674 G>A及c.673 C>T的表达载体转染的两组细胞,在胞浆胞核内均表达绿色荧光。结论不同的突变位点可以造成RUNX2蛋白亚细胞定位的改变,蛋白在核内累计表达量不足可能是造成单倍体不足的原因之一。Objective To observe the effect of the RUNX2 gene mutations on subnuclear localization of the RUNX2 mutant protein by transfection.Methods Total RNA was extracted and reverse-transcribed from blood of a patient with cleidocranial dysplasia,and the eukaryotic expression vector pEGFP-C1-RUNX2 was constructed by subcloning the objective fragments.The subnuclear localization of RUNX2 wild and mutant protein was observed by transfected into NIH3T3 fibroblast cells.Results The vector pEGFP-C1-RUNX2 was constructed and identified successfully.The results of transfection showed that both c.674 GA and c.673 CT mutation sites can contribute to partial loss of nuclear localization regardless of presence of intact nuclear localization signal,causing dual localization to both the cytoplasm and nucleus,and the wild RUNX2 protein was observed in nuclear exclusively.Conclusion Different mutation sites of RUNX2 gene can contribute to change of subnuclear localization of RUNX2 protein,and the deficiency in nuclear might be responsible for the dysfunction of RUNX2 protein in pathology of cleidocranial dysplasia.

关 键 词：RUNX2基因 突变 亚细胞定位 颅骨锁骨发育不良 

分 类 号：Q343[生物学—遗传学]