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目的构建针对细胞内氯通道4(CLIC4)的短链干涉RNA(siRNA)及其表达载体,转染细胞后观察其对CLIC4基因的干涉作用。方法设计CLIC4靶向的发夹状siRNA,据此设计合成两条互补的寡核苷酸链,退火后连接到pSilencer3.1-H1载体,转化扩增后进行序列测定。用脂质体转染法转染大鼠神经胶质瘤细胞C6,通过RT-PCR检测CLIC4基因表达水平的变化。结果酶切鉴定与DNA测序分析证明CLIC4基因的siRNA载体构建正确,RT-PCR结果表明CLIC4基因的表达水平明显降低。结论成功地构建了针对CLIC4基因的siRNA载体,转染细胞后可显著抑制CLIC4基因表达。
Objective To construct a short interfering RNA (siRNA) directed against intracellular chloride channel 4 (CLIC4) and its expression vector. The transfected cells were observed for their interference with CLIC4 gene. Methods CLIC4 targeted hairpin siRNAs were designed and synthesized. Two complementary oligonucleotide strands were designed and synthesized. After annealed, they were ligated to pSilencer3.1-H1 vector, and were sequenced after transformation and amplification. The C6 glioma cells were transfected by lipofectamine and the expression of CLIC4 gene was detected by RT-PCR. Results Enzyme digestion and DNA sequencing proved that CLIC4 gene siRNA vector was constructed correctly, RT-PCR results showed that CLIC4 gene expression was significantly reduced. Conclusion The siRNA vector targeting CLIC4 gene was successfully constructed and the expression of CLIC4 gene was significantly inhibited after transfection.