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

作　　者：李卫星[1] 杜军[2] 胡圳圳[3] 

机构地区：[1]泰州职业技术学院医学技术学院,江苏泰州225300 [2]南京医科大学基础医学院生理学系,江苏南京210029 [3]南京医科大学第二附属医院中心实验室江苏南京210011

出　　处：《南京医科大学学报（自然科学版）》2011年第11期1587-1592,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：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(2010NJ-MUZ35);中国博士后科学基金(2011M501254)

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。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.

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 

分 类 号：Q786[生物学—分子生物学]