STAT1特异性siRNA真核表达载体的构建及体外转染优化  

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR OF SIRNA SPECIFIC FOR STAT1 GENE AND OPTIMIZATION OF ITS TRANSFECTION EFFICIENCY IN VITRO

作　　者：贾晓琴[1] 熊瑛[1] 王宋平[2] 李平[1] 

机构地区：[1]泸州医学院附属医院呼吸内科,四川泸州646000 [2]泸州医学院附属医院炎症与变态反应实验室,四川泸州646000

出　　处：《泸州医学院学报》2009年第2期116-121,共6页Journal of Luzhou Medical College

基　　金：四川省卫生厅科研课题(No:070235)

摘　　要：目的:构建STAT1特异性siRNA真核表达载体,并转染人气道上皮细胞(16HBE)。方法:根据GeneBank数据库提供的STAT1基因两种变异体的共同核苷酸序列,按照Tuschl设计原则,选择设计双链siRNA,再转化为能表达其小发卡结构RNA(small hairpin RNA,shRNA)的DNA序列,并将其连接到带有卡那霉素抗性和增强绿色荧光蛋白的pGenesil-1.1真核表达载体上,将其转染至气道上皮(16HBE)细胞中,观察荧光表达,鉴定其转染效率。结果:通过酶切鉴定和序列测定,成功地构建了二条表达小干扰RNA的质粒及其阴性对照质粒,并成功转染到(16HBE)细胞株中,质粒/脂质体为1μg:2μl条件,能有效地转染细胞,转染效率达到50%以上。结论:成功构建siRNA真核表达载体并能成功体外转染,为下一步气道炎症性疾病的基因沉默研究奠定了良好的基础。Objective： To construct eukaryotic expression vector of siRNA specific for STAT1 gene and transfect into human airway epithelial cells （16HBE）. Methods： Genome sequences of STAT1 gene was retrieved from genebank,siRNA （small interfering RNA）was designed according to the Tuschl＇s principle,and was converted into cDNA coding expression of shRNA （small hairpin RNA） of siRNA for STAT1 gene. The cDNA was synthesized and inserted into pGenesil-1.1 eukaryotic expression vector with kanamycin resistance and enhanced green fluorescent protein, and then was transfected into human airway epithelial （16HBE） cells.The fluorescence expression was observed and the transfection eficiency was identified. Results： Two siRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into 16HBE cell successfully,and the transfection efficiency achieved about 50% when plasmid / lipofectamine was in 1ug：2ul conditions. Conclusion： The successful construction of recombinant STAT1-siRNA eukaryotic vectors and transfection of them into cell llne established a favourable foundation for further study of gene silencing of the airway inflammatory disease.

关 键 词：STAT1基因 小干扰RNA 小发卡结构RNA 转染 RNA干扰 

分 类 号：R562.2[医药卫生—呼吸系统] R349.5[医药卫生—内科学]