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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 a small interfering RNA (shRNA) designed to interfere with the expression of BRCA1 and cloned into pLKO.1 lentiviral vector to provide a basis for studying the role of BRCA1 in breast cancer cell proliferation. Methods: According to human BRCA1 gene sequence, three pairs of BRCA1 interference fragments were designed and synthesized (EcoRI and AgeI were added before and after the sequence), and inserted into the lentiviral vector pLKO.1 by restriction enzyme ligation. After identification and sequencing, the recombinant plasmids were transfected into MCF-7 cells. After 48h, the protein and RNA were extracted. The protein expression of BRCA1 was verified by Western blotting. The expression of BRCA1 mRNA was verified by Realtime PCR. Results: The recombinant plasmids were completely verified by restriction enzyme digestion and sequencing. The expression of BRCA1 was significantly down-regulated after transfected into breast cancer cells 48h. CONCLUSION: The construction of BRCA1-interfering lentiviral vector and the successful transfection of MCF-7 cells confirmed that BRCA1 can down-regulate the expression of BRCA1, which lays the foundation for the further study on the function of BRCA1 in breast cancer cells.