文章摘要
徐 辰,韩 晓.Ets-1重组腺病毒的构建与活性鉴定[J].南京医科大学学报,2014,(10):1436~1440
Ets-1重组腺病毒的构建与活性鉴定
Mouse Ets-1 recombinant adenovirus construction and identification
投稿时间:2014-06-11  
DOI:10.7655/NYDXBNS20141038
中文关键词: Ets-1  腺病毒  胰岛β细胞
英文关键词: acute myeloid leukemia  relapse/refractory  demethylation
基金项目:国家自然科学基金重点项目(81130013)
作者单位
徐 辰 南京医科大学江苏省人类功能基因组重点实验室,江苏 南京 210029 
韩 晓 南京医科大学江苏省人类功能基因组重点实验室,江苏 南京 210030 
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中文摘要:
      目的:构建小鼠成红细胞白血病病毒E26癌基因同系物1(V-Ets avian erythroblastosis virus E26 oncogene homolog 1,Ets-1)重组腺病毒,并验证其对小鼠原代胰岛及胰岛β细胞系INS-1的感染活性和Ets-1蛋白表达活性?方法:以小鼠胰岛β细胞系MIN6的cDNA为模板,PCR扩增Ets-1基因的编码区序列(coding sequence,CDS)区,纯化后经限制性内切酶切割和连接反应插入到pAdTrack-CMV穿梭质粒上,构建成pAdTrack-CMV-Ets-1质粒?将该质粒与腺病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中进行同源重组,鉴定出阳性克隆?扩增并抽提阳性克隆质粒,用PacⅠ酶切线性化后转染293A细胞,经包装得到AdV-Ets-1重组腺病毒?相应对照病毒AdV-GFP由相同方法构建?腺病毒经纯化后,感染小鼠原代胰岛及INS-1细胞,并用Western blot检测Ets-1蛋白表达水平?结果:腺病毒构建成功,并具有高感染率及高蛋白表达能力?结论:成功构建了小鼠Ets-1腺病毒,为进一步研究Ets-1基因在原代胰岛中的功能提供基础?
英文摘要:
      Objective:To construct a recombinant adenovirus that express mouse Ets-1 in primary cultured mouse islets. Methods:To generate pAdTrack-CMV-Ets-1 shuttle vector,the CDS region of Ets-1 was amplified by PCR,and then purified and cloned into the pAdTrack-CMV. pAdTrack-CMV-Ets-1 was recombined with back-bone pAdEasy-1 in BJ5183 bacteria. The resulting vector was transfected into QBI-293A cells to generate recombinant adenovirus. After been amplified and purified,the recombinant adenovirus were used to infect primary cultured mouse islets and INS-1 cells. The protein levels of Ets-1 were determined by Western blotting assay. Results:The Adv-Ets-1 was established successfully and proved to be high infective and possess a high expression potential. Conclusion:The Ets-1 recombinant adenovirus was successfully constructed,which provided a foundation for further study of the function of Ets-1 in primary islets.
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