摘要
目的构建AFP增强子调控的小干扰RNA质粒载体。方法从HePG2细胞中提取AFP基因。采用定向克隆的方法，构建质粒pGenesil—AFP。pGenesil—AFP质粒DNA扩增、提取后转化到DH5感受态细胞。针对目的基因Survivin的序列，构建pGenesil—AFP—shRNA。结果pGen—esil—AFP—shRNA质粒经XbaI和HindIII双酶切鉴定，获得一856bp大小和4506bp大小的片段，通过基因测序分析，证明质粒载体构建正确，用紫外分光光度计测得质粒的浓度为1．3g／L，A值为1．94，说明制备的质粒纯度较高。结论成功构建AFP增强子调控的Survivin shRNA融合质粒。
Objective To construct an AFP enhancer-regulated siRNA expression plasmid vector. Methods The AFP gene was extracted from HePG2 cells, constructing plasmid PGenesil-AFP by orientation cloned. After DNA extraction and amplification, Plasmid pGenesil-AFP was transformed into competent cell of DHS. Constructing plasmid vector pGenesil-AFP-shRNA to aim at the target gene sruvivin＇ s sequence. Results After plasmid pGenesil-AFP-shRNA evaluated by gene sequencing analysis and en- zyme cutting with both XbaI and Hind III, obtain a fragment of 856 bp and a fragment of 4506 bp. It is prove that Plasmid can be successfully constructed. Measured by ultraviolet spectrophotometer, the concentration of plasmid is 1.3 g/L and A value is 1.94. This shows that the purity of plasmid is high. Conclusion The successful construction of an AFP enhancer-regulated survivin shRNA integration plasmid.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第8期1002-1003,共2页
Chinese Journal of Experimental Surgery
基金
湖南省科技厅资助项目（2007JT2005）
关键词
癌
肝细胞
甲胎蛋白
小干扰RNA
质粒载体
Carcinoma,hepatocellular
AFP
Small interfering RNA
Plasmid vector
