电离辐射致线粒体DNA 4977 bp缺失质粒构建及其鉴定  

Construction and confirmation of the plasmid of human mitochondrial DNA 4977 bp deletion induced by ionizing radiation

作　　者：陈晓穗[1] 封江彬[2] 周丽君[1] 陆雪[2] 王欲晓[1] 陈德清[2] 曲佳[1] 刘青杰[2] 

机构地区：[1]海军总医院,北京100037 [2]中国疾病预防控制中心辐射防护与核安全医学所

出　　处：《中华放射医学与防护杂志》2006年第1期52-54,共3页Chinese Journal of Radiological Medicine and Protection

基　　金：北京市自然科学基金资助项目(7053073)

摘　　要：目的建立电离辐射诱导的人线粒体DNA(mtDNA)4977 bp缺失片段和对照DNA片段的稳定质粒,用于线粒体基因组相关研究。方法对无mtDNA 4977 bp缺失的正常人外周血进行离体照射10 Gy60Coγ射线,提取细胞总DNA,用巢式PCR扩增mtDNA 4977 bp缺失片段,普通PCR扩增对照ND1基因片段。PCR产物经纯化后构建质粒;提取质粒DNA,DNA样品经酶切、纯化后测序,将测序结果进行BLAST分析。结果PCR扩增的跨mtDNA 4977 bp缺失的DNA片段和ND1基因片段大小与预期值是吻合的。对制备的质粒DNA样品进行测序,结果经BLAST分析,两种质粒DNA与人线粒体序列同源性在99%以上。结论本研究中所构建的质粒是成功的,可以用于人mtDNA4977 bp缺失的定性或定量研究。Objective To construct a stable plasmid that spanning deleted human mitochondrial DNA （mtDNA） 4977 bp induced by ionizing radiation and another one for control DNA fragment, in order to use in the human mitochondrial genome study in the future. Methods The peripheral blood, which had no mtDNA 4977 bp deletion found in previous study, was exposed to 10 Gy ^60Co γ-rays in vitro. The total cell DNA was extracted and PCR was carried out： a nest-PCR of three-round PCR was used for the mtDNA 4977 bp deletion and oneround regular PCR was used for the control NDI： gene. The PCR products were used for transfection by electroporation and the positive clones were obtained after screening. The plasmid DNA was isolated and sequenced after enzymatic digestion and purification. The sequence result was BLASTed with the human mitochondrial genome. Results The sizes of PCR products for the flanked 4977 bp deletion and the ND1 gene were similar with those predicted according to GeneBank. The sequences for the positive clones were above 99 per cent homologous with the human mitochondrial genome after BLASTed. Conclusion The plasmids for deleted human mtDNA 4977 bp and control DNA fragment have been constructed successfully, and they could be used in the quality and quantity studies on human mtDNA 4977 bp deletion.

关 键 词：电离辐射 线粒体DNA 4977 bp缺失 质粒构建 鉴定 

分 类 号：R346[医药卫生—基础医学]