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    靶向mdr1基因shRNA真核表达载体的构建及鉴定

    Construction and identification of pSilencerTM 3.1-H1 neo mdr1 short hairpin RNA expression vectors

      摘要
    • 摘要: 目的: 设计并构建靶向mdr1基因shRNA真核表达质粒。方法: 以mdr1为靶基因设计具有短发夹结构的模板寡核苷酸,退火形成互补双链结构,再克隆至pSilencerTM3.1-H1neo载体,构建重组的短发夹shRNA表达载体。采用酶切法和测序法鉴定。结果: 经限制性内切酶酶切电泳后显示有66bp模板寡核苷酸片段和4.3kbp的线性化的pSilencerTM3.1-H1neo载体片段。重组子测序结果与Genebank中的mdr1基因cDNA序列相符。结论: 重组靶向mdr1基因shRNA表达载体构建正确,为进一步转染耐药的肿瘤细胞逆转肿瘤耐药研究奠定了基础。

       

      Abstract: Objective: To study the construction of pSilencerTM 3.1-H1 neo mdr1 short hairpin RNA expression vectors.Methods: Two different shRNA targets desigened to be homologous to the P-glycoprotein (P-gp) encoding mdr1 mRNA consensus sequence, were annealed and ligated into the BamHⅠ and Hind Ⅲ site of linearized pSilencerTM 3.1-H1 neo vector.The recombinant named as pSilencerTM3.1-H1 neo mdr1-A and mdr1-B shRNA expression plasmids was identificated by restrictive enzyme digestion and sequencing.Results: The fragments of 66 bp and 4.3 kbp were shown after digestion by BamHⅠ and Hind Ⅲ and agarose gel electrophoresis.The DNA sequences of pSilencerTM3.1-H1 neo mdr1-A and mdr1-B shRNA expression plasmids were proved to be identical to the data of mdr1 in Genebank.Conclusions: pSilencerTM3.1-H1 neo mdr1 shRNA expression vectors has been constructed successfully.

       

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