人BRCA1干涉慢病毒载体的构建与验证

Construction and Verification of Lentiviral Vectors Targeting for Human BRCA1 Gene

目的:设计合成干涉BRCA1表达的小干扰RNA,并克隆入pLKO.1慢病毒表达载体,为研究基因BRCA1在乳腺癌细胞增殖中的作用提供基础。方法:根据人BRCA1的基因序列,设计合成三对BRCA1干涉片段(序列前后加入酶切位点EcoRI和AgeI),再利用酶切连接反应将其插入到慢病毒载体pLKO.1中,经过酶切鉴定及测序正确后,将重组质粒转染入MCF-7细胞,48h后提取蛋白质和RNA,通过蛋白印迹验证BRCA1的蛋白水平的表达情况,Realtime PCR验证BRCA1的RNA水平的表达变化。结果:重组质粒经酶切鉴定和测序比对完全正确,转染乳腺癌细胞48h后可见BRCA1表达的明显下调。结论:成功构建BRCA1干涉的慢病毒载体,并且转染MCF-7细胞证实其能够下调BRCA1的表达,为后续研究BRCA1在乳腺癌细胞的功能奠定了基础。

Objective： To design and synthesize three small interference RNA targeting for human BRCA1 gene,and translate them into a lentiviral expression vector pLKO.1,lay the foundation for further study of BRCA1 functions in breast tumor cells.Methods： To construct the lentiviral vectors of small interference RNA targeting for BRCA1 gene.Three interference fragments were designed and synthesized based on human BRCA1 gene sequence,and then they were digested by double restriction enzymes and inserted into the lentiviral expression vector pLKO.1.After confirmation by enzyme digestion and sequencing,the constructed plasmids were transfected into MCF-7 breast tumor cells.48 hours later,BRCA1 expression were verified by Western-blot and real-time PCR.Results：Three small lentiviral plasmids were all 500 bp.The constructed recombination plasmids were verified by enzyme digestion and DNA sequencing.BRCA1 gene expression can be down-regulated in MCF-7 cells after transfection.Conclusion： The lentiviral vectors with siRNAs targeting human BRCA1 gene were successfully constructed,and they was able to down-regulate BRCA1 gene expression significantly in MCF-7 cells after cell transfection,which lays the foundation for the following studies.