PYGO_2基因RNA干扰真核表达载体的构建  

Construction of PYGO2 eukaryotic expression vector for RNA interference

作　　者：王海东[1] 苏新辉[2] 王占祥[1] 马永会[1] 谭国伟[1] 

机构地区：[1]厦门市第一医院神经外科,福建厦门361006 [2]厦门市第一医院核医学科,福建厦门361006

出　　处：《中华神经外科疾病研究杂志》2009年第2期127-130,共4页Chinese Journal of Neurosurgical Disease Research

摘　　要：目的构建PYGO2基因RNA干扰(RNAi)的真核细胞表达载体。方法以PYGO2为靶基因,以pSUPER.puro质粒为载体,设计构建重组体,根据基因库(GenBank)提供的PYGO2基因核苷酸序列,在http://www.ambion.com网站上使用siRNA Target Finder软件进行目的基因的siRNA序列对应DNA的设计与筛选,选择设计两条带发夹结构的核苷酸序列,克隆到空载体pSUPER.puro中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析,将重组的pSUPER.puro-PYGO2质粒转染胶质瘤C6细胞48h,检测其对该细胞PYGO2蛋白的影响。结果经酶切鉴定筛选出的重组体测序结果与目的序列完全一致,重组载体显著降低胶质瘤细胞PYGO2的蛋白表达,重组载体构建成功。结论利用RNAi技术可成功构建抑制PYGO2表达的siRNA真核表达载体。Objective To construct PYGO2 eukaryotic expression vector for RNA interference. Methods Recombinant were designed and established by targeting gene PYGO2 and plasmid pSUPER, puro. Based on PY- GO2cDNA sequences of genomes, two pairs of oligonucleotides were synthesized by on-line Target Finder RNAi design software and inserted into plasmid pSUPER, puro to generate siRNA eukaryotic expression vector. DH5 α strains were transformed, plasmid were extracted and recombinant vector were identified by the restriction map and sequence analysis. The recombinant pSUPER, puro- PYGO2 was transfected into the cultured C6 cells. At 48 h after transfection, the whole cell proteins were extracted and the protein level was detected by Western blot with goat-anti-mouse PYGO2 monoclonal antibody. Results Recombinant plasmids completely matched the outcome by the restriction map and the sequence analysis, pSUPER, puro- PYGO2 expression vector into C6 ceils down-regulated the protein level of PYGO2 at 48 h after transfection and the recombinant eukaryotic expression vectors were constructed successfully. Conclusion siRNA recombinant plasmid can be constructed successfully by RNAi technique for inhibition of PYGO2 expression.

关 键 词：PYGO2 RNA干扰 真核表达载体 构建 

分 类 号：R346[医药卫生—基础医学]