作者：李发武 卢放根 余治健 吴福全 王文琦 Fa-...靶向性血小板衍生生长因子sirna表达质粒克隆构建测序鉴定growthfactorsmallinterferingrnagelelectrophoresis测序结果基因功能插入片段interference琼脂糖凝胶电泳genesequence转化大肠杆菌小分子干扰序列设计合成酶切位点
摘要：目的 构建靶向性血小板衍生生长因子(PDGF)B链的小分子干扰RNA(siRNA)表达质粒,为进一步研究PDGF基因功能奠定基础.方法 根据PDGF-B链序列设计合成含靶向PDGF基因siRNA转录模板的茎环结构,与两端分别有Bam HI、HindIII酶切位点的pSilencer3.1-Hlhygro质粒连接,转化大肠杆菌,扩增、纯化得到所需质粒,通过琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列.结果 PCR扩增片段与预期结果相符,双酶切证实PDGF siRNA表达载体克隆构建成功,插入片段测序结果与合成的siRNA结果一致.结论 成功构建了靶向性PDGF基因的siRNAs表达质粒. Abstract： Objective To construct the small interfering RNA (siRNA) expressing plasmids targeting platelet-derived growth factor(PDGF)and study the function of PDGF with RNA interference technology. Methods Two complementary 63-nt oligonucleotides targeting PDGF were synthesized with 5 single-stranded over-hangs according to the PDGF-B gene sequence in the Genebank and the kit manual,which were ligated with the linearized pSilencer3.1-Hlhygro.The plasmids were transformed into DH5α bacteria to amplify and then purified.The purified plasmids were identified by gel electrophoresis and sequencing.Results PDGF siRNA expression vectors were successfully constructed and identified by double endonuclease digestion.Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonucleotides.Conclusions siRNA expression plasmids targeting PDGF have been successfully constructed.
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