MAGE3 siRNA表达载体的构建及其沉寂作用  

Vector Construction and Silencing Effect of MAGE3 Gene Targeted Small Interfering RNA

作　　者：李玲[1] 李怀洲[2] 王华启[3] 王蕾[2] 张国俊[3] 杨胜利[2] 赵国强[2] 

机构地区：[1]郑州市骨科医院骨肿瘤科,河南郑州450052 [2]郑州大学基础医学院,河南郑州450052 [3]郑州大学第一附属医院呼吸科,河南郑州450052

出　　处：《河南肿瘤学杂志》2005年第6期384-386,共3页Henan Journal of Oncology

基　　金：河南省医学创新人才项目(20043008)

摘　　要：目的构建针对人MAGE3基因的小干扰RNA(siRNA)及其表达载体,转染细胞后观察其对MAGE3基因的干扰作用。方法设计MAGE3靶向的发夹状siRNA,依据设计合成两条互补的寡核苷酸链,退火后连接入pSUPER.neo载体,转化扩增后进行序列测定。用脂质体包裹转染胃癌细胞BGC823,采用RT-PCR检测MAGE3基因mRNA表达水平的变化。结果把针对MAGE3基因的siRNA的双链寡核苷酸片段克隆到pSUPER.neo载体,经过酶切鉴定与测序,结果正确;稳定转染人胃癌细胞BGC823后,RT-PCR检测显示,MAGE3基因的表达水平明显降低。结论成功构建了针对MAGE3基因的siRNA载体,转染细胞后可抑制MAGE3基因表达。Objective To construct MAGE3 gene-targeted small interfering RNA（siRNA） and its expression vector; and to observe the expression level of its expression vector MAGE3 gene in human gastric carcinoma BGC823 cell transfected by the vector. Methods First to design MAGE3 gene-targeted hairpin siRNA, then to synthesize two complementary oligo nucleotide strand, after annealing, to insert the two-strand oligo nucleotide into pSURER neo vector , which was then restrictive enzyme digested and sequenced. After that we transfected human gastric carcinoma BGC823 cell line with the vector using lipofectamine method. Finally, detected the mRNA expression level of MAGE3 gene through RT-PCR. Results Restric- tive enzyme digestion and sequencing confirmed the vector containing siRNA was what we wanted ; RT-PCR showed the expression level of MAGE3 gene in transfected BGC823 was decreased. Conclusion We had constructed the correct MAGE3 gene targeted siRNA and its vector, and the vector could obviously reduce the MAGE3 gene expression after transfecting.

关 键 词：RNA干扰 MAGE3 表达 

分 类 号：R730.231[医药卫生—肿瘤]