文章摘要
李卫星,杜 军,胡圳圳.pEGFP-N1/Arf6-T27N重组真核表达载体的构建及其在人乳腺癌细胞株中的表达[J].南京医科大学学报,2011,(11):1587~1592
pEGFP-N1/Arf6-T27N重组真核表达载体的构建及其在人乳腺癌细胞株中的表达
Construction of the recombinant eukaryote expression vector carrying encoding gene of Arf6-T27N and its expression in breast cancer cells
投稿时间:2011-08-08  
DOI:10.7655
中文关键词: Arf6-T27N  MDA-MB-231人乳腺癌细胞  基因转染  稳定表达  细胞迁移
英文关键词: Arf6-T27N  MDA-MB-231 breast cancer cell  gene transfection  stable expression  cell migration
基金项目:南京医科大学基础医学院青年教师培养基金(2010);南京医科大学科技发展基金重点项目(2010NJMUZ35);中国博士后科学基金(2011M501254)
作者单位
李卫星 泰州职业技术学院医学技术学院,江苏 泰州 225300 
杜 军 南京医科大学基础医学院生理学系,江苏 南京 210029 
胡圳圳 南京医科大学第二附属医院中心实验室 江苏 南京 210011 
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中文摘要:
      目的:构建含有Arf6-T27N基因的重组真核表达载体,并检测Arf6-T27N蛋白在MDA-MB-231人乳腺癌细胞中的表达情况,以期为深入研究Arf6在乳腺癌细胞迁移中的作用机制奠定基础?方法:从含Arf6-T27N基因的真核表达质粒pXS-Arf6-T27N中扩增出Arf6-T27N基因,插入pEGFP-N1载体中构建成pEGFP-N1/Arf6-T27N,利用脂质体将其转染MDA-MB-231人乳腺癌细胞?用新霉素筛选稳定表达的细胞系,Western blot确定重组蛋白表达,并用Boyden chamber小室实验检测转染Arf6-T27N的MDA-MB-231人乳腺癌细胞迁移能力的改变?结果:限制性内切酶鉴定和核酸定列测定证实成功构建了含Arf6-T27N的重组真核表达载体pEGFP-N1/Arf6-T27N?以重组质粒稳定转染MDA-MB-231人乳腺癌细胞,能检测到Arf6-T27N蛋白的表达,且Arf6-T27N蛋白能明显抑制MDA-MB-231人乳腺癌细胞的迁移?结论:成功构建了重组质粒pEGFP-N1/Arf6-T27N,稳定转染Arf6-T27N的MDA-MB-231人乳腺癌细胞可检测到其蛋白水平的稳定表达,重组质粒介导的Arf6-T27N蛋白能明显抑制MDA-MB-231细胞的迁移?
英文摘要:
      Objective: To construct the recombinant eukaryote expression vector containing Arf6-T27N gene and detect the protein expression in its target cell line,MDA-MB-231. Methods: The fragment of Arf6-T27N gene from expression vector pXS-Arf6-T27N was cloned into the eukaryote expression vector pEGFP-N1,then the recombinant vector pEGFP-N1/Arf6-T27N were transfected into the MDA-MB-231 cells. After transfection of the recombinant vector,the protein expression of Arf6-T27N in MDA-MB-231 cells were detected by Western blot. The effect of Arf6-T27N on cell migration was investigated by Boyden chamber assay. Results: The recombinant eukaryote expression vector carrying Arf6-T27N was constructed successfully. The expression of Arf6-T27N in MDA-MB-231 cells could be detected by Western blot. In addition,Arf6-T27N protein could inhibit the migration of MDA-MB-231 cells. Conclusion: The new recombinant expression vector pEGFP-N1/Arf6-T27N was constructed successfully and the MDA-MB-231 cells which could express Arf6-T27N stably were also successfully made. Furthermore,our study suggested that Arf6-T27N protein could inhibit the migration of MDA-MB-231 cells.
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