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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。
Objective To construct an RNA interference (RNAi) recombinant lentiviral vector containing cytochrome P450 (CYP) 4F3 gene. Methods The CYP4F3 siRNA sequence was designed and synthesized, ligated with pGCL vector containing U6 promoter and green fluorescent protein (GFP) to generate PscsiCYP4F3 lentiviral vector, and identified by PCR and sequencing. 293T cells were co-transfected with 293T cells with PscsiCYP4F3, pHelper1.0 and pHelper2.0 plasmids to produce lentivirus, and the virus titer was determined. The expression of CYP4F3 mRNA in human lung adenocarcinoma A549 cells was determined to be effective in interfering with this gene Targets. Results The lentiviral vector of CYP4F3siRNA was successfully constructed and the target of effective interference was screened out. Conclusion The RNAi lentiviral vector of human CYP4F3 gene was successfully constructed, which laid the foundation for further studies on CYP4F3 gene function.