抑制HIF-1α表达的siRNA序列长效shRNA抑制载体的构建  

Construction of shRNA expression vector for silencing HIF-1α inducible protein expression

作　　者：郭欢[1,2] 孙蕾[1,2] 汲翔[2] 康巧珍[2] 程道博[1] 张聚真[1] 赵立群[1] 杨观瑞[1] 汲振余[1,2] 

机构地区：[1]郑州大学医药科学研究院肿瘤室,郑州450052 [2]郑州大学生物工程系肿瘤生物学多肽生物化学教研室,郑州450001

出　　处：《郑州大学学报（医学版）》2011年第2期173-176,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30873002;30972707

摘　　要：目的:设计特异抑制缺氧诱导因子-1α(HIF-1α)蛋白诱导表达的小干扰RNA(siRNA)序列,并构建能长效抑制HIF-1α基因表达的HIF-1α的短发夹siRNA(shRNA)表达载体。方法:依据NCBI数据库获得HIF-1αmR-NA序列,利用Whitehouse、SiDirect和Rationalsi RNA Design软件及Blast设计和优化siRNA序列;将siRNA序列转换成编码shRNA的DNA序列后克隆入pENTRTM/H1/TO载体,测序鉴定;将重组质粒pENTRTM/H1/TOHIF-1α转染食管上皮细胞Het-1A(实验组),并设阴性对照(转染阴性对照序列)、空白对照组和诱导对照组。实验、阴性对照和诱导对照组细胞经CoCl2化学诱导。Western blot法检测4组HIF-1α蛋白的表达。结果:以优化改造的含29核苷酸的干扰序列构建shRNA表达载体,经测序鉴定无误。4组细胞HIF-1α蛋白表达比较,差异有统计学意义(F=66.863,P<0.001)。实验组Het-1A细胞HIF-1α蛋白表达较诱导对照和阴性对照组明显下降。结论:成功构建了HIF-1αshRNA表达载体。Aim：To predict the small interfering RNA（siRNA） sequence using the bio-informatics technology and construct short hairpin RNA（shRNA） expression vector for long-term inhibition of hypoxiable inducible factor-1α（HIF-1α）.Methods：According to the HIF-1α sequence obtained from NCBI database,Whitehouse,siDirect and Rational siRNA Design software were used to predict the siRNA interference sequence and followed by homological analysis with Blast.DNA oligos encoding shRNA were synthesized and cloned into pENTRTM/H1/TO vector followed by identification.pENTRTM/H1/TOHIF-1α recombinant plasmid was transfected into esophageal epithelial cell line Het-1A.HIF-1α protein expression was examined by western blot.Results：An optimum sequence with 29 nucleotides were obtained.shRNA expression vector was constructed with the 29 nucleotides fragment and identified by sequencing.The expression of HIF-1α protein among four groups were different,there were obvious differences（F=66.863,P0.001）.The chemical induction made HIF-1α protein expression decreased significantly.Conclusion：HIF-1α shRNA interfere expression vector has been successfully constructed.The bio-informatics technology could be conveniently applied to for HIF-1α siRNA design which can promote the further study.

关 键 词：缺氧诱导因子-1Α 小干扰RNA 短发夹RNA Het-1A细胞 

分 类 号：R735.1[医药卫生—肿瘤]