钙转运蛋白基因siRNA表达载体的构建及鉴定  

Construction and identification of siRNA expression vectors for silencing Ca^(2+) transport protein gene

作　　者：杨新宇[1] 张雷[2] 郑花[2] 康劲松[2] 李洪岩[2] 房学东[1] 

机构地区：[1]吉林大学第二临床医院,长春130041 [2]吉林大学白求恩医学院病理生理教研室,长春130021

出　　处：《中国免疫学杂志》2008年第3期257-259,262,共4页Chinese Journal of Immunology

摘　　要：目的:构建针对钙转运蛋白(CaT1)特异性干扰RNA(siRNA)及其表达载体,并检测其对CaT1基因的干扰作用。方法:设计CaT1靶向的发夹状siRNA,合成两条互补的寡核苷酸链,退火后连接入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人类胃癌细胞BGC,通过RT-PCR检测CaT1基因表达水平的变化。结果:酶切鉴定与DNA测序分析证明CaT1基因的siRNA载体构建正确,RT-PCR结果表明CaT1基因的表达水平明显降低。结论:成功地构建了针对CaT1基因的siRNA载体,转染细胞后可抑制CaT1基因表达。Objective： To construct specific small interfering RNA（siRNA） expressing vectors of intracellular Ca^2＋ transport protein （CaT1）gene and detect its silencing effects.Methods： The hairpin sequences of siRNAs targeting CaT1 gene were designed, synthesized and cloned into pSlincer 3.1-HI plasmids after annealing. The vectors were then enriched in E. coli. The recombinant pSlincer 3.1-HI plasmids were identified by restriction endonuclease cutting and DNA sequencing and then transfected into Human Gastric Carcinoma Cell Line BGC- 823.The expression of CaT1 mR.NA was examined by RT-PCR. Results： The siRNA oligonucleotides of CaT1 were correctly cloned into the pSlincer 3.1-HI plasmids and confirmed by restriction endonuclease cutting and DNA sequencing. RT-PCR analysis revealed that the expression of CaT1 mR.NA in BGC-823 cells transfected with the pSlincer 3.1-HI constructs of siRNA was significantly decreased compared with that of the negative control and untransfected group. Conclusion： siRNA expression plasmids for silencing Ca^2＋ transport protein gene are successfully constructed, and they effectively inhibit the CaT1 gene expression.

关 键 词：SIRNA RNA干扰 钙转运蛋白 人胃癌细胞株BGC-823 

分 类 号：R730[医药卫生—肿瘤]