甲胎蛋白增强子调控的小干扰RNA质粒载体的构建

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Hind Ⅲ双酶切鉴定,获得一856 bp大小和4506 bp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1.3 g/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.

Construction of an AFP enhancer-regulated siRNA expression plasmid vector

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, Plasrnid pGenesil-AFP was transformed into competent cell of DHS.Constructing plasmid vector pGenesil-AFP-shRNA to aim at the target gone sruvivin' s sequence.Results After p]asmid pGenesil-AFP-shRNA evaluated by gene sequencing analysis and enzyme 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.