携带DREAM基因小分子干扰RNA重组腺相关病毒包装质粒pSANV2.0的构建

[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。

Construction of Packaging Plasmid pSANV2.0 of Adeno-associated Virus Vector(AAV) Carrying DREAM-targeting siRNA

[Purpose] To construct the packaging plasmid pSANV2.0-DREAMshRNA-EGFP of adeno-associated virus vector(AAV)carrying DREAM-targeting siRNA(small interfering RNA),which provide foundation for further study on the effect of DREAM gene for cancer pain treatment.[Methods ] We designed and synthesized two complementary oligonucleotide strands containing the small hairpin of DREAM,which was inserted into the EcoRⅠ and SalⅠ sites of pDC316-EGFP-U6 plasmid double digested by EcoRⅠ and SalⅠ.We got resultant plasmid pDC316-EGFP-DREAMshRNA-U6 and used it serving as PCR template to propagate DREAMshRNA-EGFP sgement,simutaneously EcoRⅠ and SalⅠ sites were introduced into PCR products.We ligated the PCR products and pSANV2.0 double digested by EcoRⅠ and SalⅠ to get resultant plasmid pSANV2.0-DREAMshRNA-EGFP.The liation product was transformed competence E.coli DH5.Positive clones were identified by PCR,enzyme digestion and sequencing.[Results] The result of PCR,restrictive enzyme digestion and gene sequencing confirmed that the inserted gene sequence and sites were correct and the plasmid pSANV2.0-DREAMshRNA-EGFP had been reconstructed successfully.[Conclusion] The packaging plasmid pSANV2.0-DREAMshRNA-EGFP of adeno-associated virus vector(AAV)carrying DREAM-targeting siRNA is successfully constructed.