CYP4F3基因RNA干扰重组慢病毒载体的构建

摘要

Objective To construct a recombinant lentiviral vector for RNA interference (RNAi) of cytochromo P450(CYP)4F3 gene. Methods Sequence of CYP4F3 siRNA was designed,synthesized, cloned into the pGCL vector containing U6 promoter and green fluorescent protein(GFP) and confirmed by PCR and sequencing. Human embryonic kidney 293T cells were co-transfected with PscsiCYP4F3 lentiviral vector, pHelperl. 0 vector andpHelper 2. 0 vector, and the lentivirus was produced. The titer of virus was detected according to the expression of GFP, and the expression of CYP4F3 mRNA in human lung adenocarcinoma A549 cells was used to determine the effective target of interference. Results The lentiviral vector for CYP4F3 siRNA was successfully constructed, and the effective target of interference for CYP4F3 gene was screened. Conclusion The lentiviral vector for RNAi of CYP4F3 gene has been successfully constructed, which provides a possibility for further investigation of its function.%目的 构建细胞色素P450(CYP)4F3基因RNA干扰(RNAi)重组慢病毒载体.方法 设计并合成CYP4F3 siRNA序列,与含U6启动子和绿色荧光蛋白(GFP)的pGCL载体连接产生PscsiCYP4F3慢病毒载体,PCR和测序鉴定.用PscsiCYP4F3、pHelper1.0和pHelper2.0质粒共转染人肾上皮细胞系293T细胞,包装产生慢病毒,测定病毒滴度,并以人肺腺癌A549细胞中CYP4F3mRNA表达水平确定该基因干扰的有效靶点.结果 成功构建CYP4F3siRNA的慢病毒载体,并筛选出该基因的有效干扰靶点.结论 成功构建人CYP4F3基因RNAi慢病毒载体,为后期研究CYP4F3基因功能奠定基础.