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目的构建BRAF基因序列特异性的短发卡RNA(shRNA)质粒载体,检测其对人黑素瘤细胞BRAF基因的干扰作用。方法设计2条BRAF基因序列特异性的shRNA编码序列和1条非特异性阴性对照序列,插入质粒pGenesil-1,双酶切和测序鉴定。用构建成功的3条质粒转染人黑素瘤细胞株A375和M14,分别采用RT-PCR和蛋白印迹检测其BRAF基因的mRNA和蛋白表达。结果所设计的shRNA序列被正确插入质粒pGenesil-1。非特异性对照质粒neg对黑素瘤细胞BRAF基因表达不产生干扰,2条序列特异性质粒braf 1和braf 2抑制了BRAF基因mRNA和蛋白的表达,其中braf 1干扰效果最为明显,最高抑制率可达90%。结论质粒介导的shRNA可成功干扰人黑素瘤细胞BRAF基因的表达,其抑制时间可持续至少1个月。
Objective To construct a short hairpin RNA (shRNA) plasmid vector with BRAF gene sequence and detect its interference with BRAF gene in human melanoma cells. METHODS: Two BRAF gene-specific shRNA coding sequences and one nonspecific negative control sequence were designed and inserted into plasmid pGenesil-1 for double enzyme digestion and sequencing. Three constructed plasmids were transfected into human melanoma cell lines A375 and M14, and the mRNA and protein expression of BRAF gene were detected by RT-PCR and Western blot, respectively. The designed shRNA sequence was correctly inserted into plasmid pGenesil-1. Non-specific control plasmid neg had no effect on the expression of BRAF gene in melanoma cells. Two sequence-specific plasmids, braf 1 and braf 2, inhibited the expression of BRAF mRNA and protein. The highest inhibition rate of braf 1 was the highest Up to 90%. Conclusion Plasmid-mediated shRNA can successfully interfere with the expression of BRAF gene in human melanoma cells, and its inhibitory time can last at least one month.