期刊论文

中文

生物医学工程学杂志

1001-5515

2014

31

5

1085-1089

10.7507/1001-5515.20140204

国家自然科学基金资助项目（81172712） 湖南省自然科学基金资助项目（11JJ6078） 湖南省教育厅优秀青年基金资助项目（09B087） 南华大学博士启动基金资助项目（2010XQD19）

本校为第一机构

为了构建pGenesil-1-Beclin1真核表达载体,并建立Beclin1稳定沉默的人成神经瘤SH-SY5Y细胞系,将合成的shRNA片段连接pGenesil-1质粒,构建成重组质粒pGenesil-1-Beclin1,转化其至感受态大肠杆菌JM109,挑取阳性克隆进行双酶切和DNA测序鉴定.常规培养SH-SY5Y细胞,将重组质粒pGenesil-1-Beclin1和空白对照质粒pGenesil-1转染SH-SY5Y细胞,G418抗性筛选,在荧光显微镜下挑取单细胞克隆并扩大培养至稳定细胞株,经RT-PCR和Western blot检测Beclin1基因的表达.经酶切和DNA测序鉴定重组真核表达载体构建正确,成功筛选出两株细胞系.和对照组转染pGenesil-1的细胞系相比,转染pGenesil-1-Beclin1细胞系的Becli...

The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E. coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were ac...