细胞内氯通道4基因siRNA表达载体的构建及其沉默效应

目的构建针对细胞内氯通道4（CLICA）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CLICA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CLICA基因表达水平的变化。结果酶切鉴定与DNA测序分析证明CLICA基因的siRNA载体构建正确，RT-PCR结果表明CLICA基因的表达水平明显降低。结论成功地构建了针对CLICA基因的siRNA载体，转染细胞后可显著抑制CLICA基因表达。

Vector construction and silencing effect of intracellular chloride channel 4-targeted siRNA

Objective To construct intracellular chloride channel 4 （CLIC4） gene--specific small interfering RNA（siRNA） expression vectors and detect their silencing effects. Methods CLIC4 mRNA targeted hairpin siRNAs were devised and the oligonucleotide strands of DNA fragments encoding the above siRNAs were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into a pSilencer 3.1-H1 plasmids, followed by amplification in E.coli and DNA sequencing. The resulting recombinant pSilencer 3.1-H1 constructs were then introduced into rat malignant astracytoma cell line（C6） and the expression of CLIC4 mRNA was examined by RT-PCR. Results The DNA fragments encoding CHC4-targeted siRNA were cloned into the pSilencer 3.1-H1 plasmids and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR analysis revealed a strongly decreased level of CLIC4 mRNA in C6 cells stably transfected with the pSilencer 3. 1-H1 comtructs of siRNA compared with the negative control or untransfected group. Conclusion The recombinant siRNA expression plasmids can significantly inhibit the CLIC4 gene expression（ P 〈 0.05）.