DNMT3 a基因shRNA质粒表达载体的构建及其沉默作用

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DNMT3 a基因shRNA质粒表达载体的构建及其沉默作

用

吴艳凤;李德涛;郑倩;李雪;薛成军;陈辉

【期刊名称】《中国老年学杂志》【年(卷),期】2015(000)006

【摘要】Objective To construct the eukaryotic expression vector carrying double shRNA sequence targeting conserved domain of DNMT3a mRNA and negative control ( HK) in pGensil-1 by using technology of gene recombination .Methods The recombinant plasmids were transformed into competent Escherichia coli DH 5α.The positive clone E.coli was screened and enriched by kanamycin (50μg/ml), and then the recombinant plasmids were extracted from the DH 5αand evaluated by restriction enzymes and sequence analysis .Cisplatin-re-sistant human lung adenocarcinoma A 549 cells ( A549-DDP) were chosen and transfected with the recombinant plasmids , semi-quantitative RT-PCR was performed to confirm the inhibitory rates of DNMT 3a mRNA before and after plasmid transfecting in 24, 48, 72 h.Results Sequence analysis showed the right sequence and the recombinant plasmids couldn 't be digested by PstⅠ, and pGenesil-1-DNMT3a-shRNA transfected could significantly decrease the DNMT 3a mRNA with the inhibitory rates of 25.5%, 56.2%, 63.4%, respectively, and the effect was dependent on the time duration .However, pGenesil-1-

HK-shRNA barely affected the expression of DNMT3a

mRNA.Conclusions The eukaryotic expression vectors are successfully constructed , which laids a foundation of further research .%目的：以DNMT3a mRNA 保守序列为靶基因设计针对DNMT3a mRNA的siRNA序列和无效干扰对照序列（HK），并利用基因重组技术将其克隆到真核表达载体pGensil－1中，构建pGenesil－1－DNMT3a－shRNA、pGenesil－1－HK－shRNA重组质粒。方法应用转化技术将重组质粒转化到大肠杆菌 DH5a中，通过卡那霉素抗性筛选出阳性菌落后提取质粒进行酶切和测序鉴定，应用质粒转染技术转染耐顺铂肺腺癌 A549细胞（ A549－DDP），利用RT－PCR技术检测转染重组质粒24、48、72 h后 DNMT3a的沉默效率。结果测序结果与合成序列一致，重组质粒不能够被PstⅠ切开，转染pGenesil－1－DNMT3a－shRNA能够明显抑制DNMT3a的表达，抑制率分别达到25．5％，56．2％，63．4％，且呈明显时间依赖性，而 pGenesil－1－HK－shRNA无明显抑制作用。结论成功构建了DNMT3a基因shRNA及HK质粒真核表达载体，为进一步实验研究奠定了基础。【总页数】4页(1612-1615)

【关键词】DNMT3a;RNA干扰;小发卡RNA;重组质粒;A549-DDP细胞株【作者】吴艳凤;李德涛;郑倩;李雪;薛成军;陈辉

【作者单位】重庆医科大学附属第一医院检验科，重庆 400016;重庆医科大学附属第一医院检验科，重庆 400016;重庆医科大学附属第一医院检验科，重庆 400016;重庆医科大学附属第一医院检验科，重庆 400016;重庆医科大学附属第一医院检验科，重庆 400016;重庆医科大学附属第一医院检验科，重庆

400016

【正文语种】中文【中图分类】R734.2 【文献来源】

https://www.zhangqiaokeyan.com/academic-journal-cn_chinese-journal-gerontology_thesis/0201231695647.html 【相关文献】

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