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[目的]构建携带DREAM基因小分子干扰RNA(siRNA)的腺相关病毒载体包装质粒pSANV2.0-DREAMshRNA-EGFP,为进一步研究DREAM基因在癌痛基因治疗中的作用奠定基础。[方法]设计并合成包含DREAM短发夹序列(shRNA)两条互补的寡核苷酸链,退火后插入到经过EcoRⅠ和SalⅠ双酶切的pDC316-EGFP-U6质粒的EcoRⅠ和SalⅠ位点,得到重组质粒pDC316-EGFP-DREAMshRNA-U6,然后以其为模板,通过PCR扩增DREAMshRNA-EGFP片段,并引入EcoRⅠ和SalⅠ位点,PCR产物与pSANV2.0经EcoRⅠ和SalⅠ酶切后连接得到重组质粒pSANV2.0-DREAMshRNA-EGFP,转化入感受态大肠杆菌DH-5α,获得阳性克隆进行PCR、酶切和测序鉴定。[结果]PCR、酶切鉴定以及测序结果证实,插入的片段序列和位点完全正确,重组质粒pSANV2.0-DREAMshRNA-EGFP的构建成功。[结论]成功构建携带DREAM基因小分子干扰RNA重组腺相关病毒包装质粒pSANV2.0-DREAMshRNA-EGFP。
[Objective] To construct adeno-associated virus vector-packaging plasmid pSANV2.0-DREAMshRNA-EGFP carrying DREAM gene small interfering RNA (siRNA), which laid the foundation for further study on the role of DREAM gene in cancer pain gene therapy. [Method] Two complementary oligonucleotide strands containing DREAM short hairpin sequence (shRNA) were designed and synthesized. After annealed, they were inserted into EcoRI and SalI sites of plasmid pDC316-EGFP-U6 digested with EcoRI and SalI, The recombinant plasmid pDC316-EGFP-DREAMshRNA-U6 was obtained. The DREAM shRNA-EGFP fragment was amplified by PCR and introduced into EcoRI and SalI sites. The PCR product was ligated with pSANV2.0 and ligated with EcoRI and SalI to obtain a recombinant The plasmid pSANV2.0-DREAMshRNA-EGFP was transformed into competent E. coli DH-5α to obtain positive clones for PCR, enzyme digestion and sequencing. [Result] PCR, restriction enzyme digestion and sequencing confirmed that the sequence and site of insert were completely correct. The construction of recombinant plasmid pSANV2.0-DREAMshRNA-EGFP was successful. [Conclusion] The recombinant adeno-associated virus packaging plasmid pSANV2.0-DREAMshRNA-EGFP carrying DREAM gene small interfering RNA was successfully constructed.