容量调控氯通道基因siRNA表达载体的构建及其沉寂效应检测  

Vector construction and silencing effect of volume-regulated chloride channel gene-targeted small interfering RNA

作　　者：张勇[1] 贾林涛[1] 李庆霞[1] 王成济[1] 周士胜[2] 杨安钢[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033 [2]大连大学医学院医学研究中心,辽宁大连116622

出　　处：《第四军医大学学报》2005年第9期769-772,共4页Journal of the Fourth Military Medical University

基　　金：国家"973"计划资助(2004CB518805);国家高科技研究与发展计划"863"资助(2004AA217071);国家自然科学基金(30270602)

摘　　要：目的:构建针对容量调控氯通道基因(CLC3)的小干扰RNA(siRNA)及其表达载体,转染细胞后观察其对CLC3基因的干扰作用.方法:设计CLC3靶向的发夹状siRNA,据此设计合成两条互补的寡核苷酸链,退火后连接入pSUPER载体,转化扩增后进行序列测定.用脂质体转染法转染胃癌细胞SGC7901,通过RT PCR、间接免疫荧光检测CLC3基因表达水平的变化.结果:把针对CLC3基因的siRNA的双链寡核苷酸片段克隆到pSUPER载体,经过酶切鉴定与测序,结果正确,稳定转染人胃癌细胞SGC7901后,RT PCR、间接免疫荧光检测表明,CLC3基因的表达水平明显降低.结论:构建了针对CLC3基因的siRNA载体,转染细胞后可抑制CLC3基因表达.AIM: To generate volume-regulated chloride channel (CLC3) gene-specific small interfering RNA (siRNA) and its expression vector. METHODS: CLC3 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 pSUPER plasmid, followed by amplification in E. coli and DNA sequencing. The resulting pSUPER constructs were then introduced into human stomach carcinoma SGC7901 cells and the expression of CLC3 gene was examined by RT-PCR and indirect immunofluorescence. RESULTS: The DNA fragments encoding CLC3-targeted siRNA were cloned into the pSUPER plasmid and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and indirect immunofluorescence analysis revealed a strongly decreased level of CLC3 mRNA and the protein in SGC7901 cells stably transfected with the pSUPER constructs of siRNA in comparison with the mock-transfected or untransfected cells. CONCLUSION: We have successfully generated an siRNA construct which can significantly inhibit the CLC3 gene expression.

关 键 词：RNA干扰 容量调控氯通道 胃肿瘤 

分 类 号：Q255[生物学—细胞生物学]