DREAM基因小分子干扰RNA重组腺相关病毒载体的构建与鉴定

目的构建表达DREAM基因的小分子干扰RNA(siRNA)重组腺相关病毒(rAAV)载体,并观察感染PC12细胞后DREAM蛋白表达受抑制情况。方法设计并合成shRNA对应的2条互补的寡核苷酸链,pDC316-EGFP-U6质粒经BamHⅠ和HindⅢ双酶切与退火后的寡核苷酸连接,构建pDC316-EGFP-DREAMshRNA-U6质粒,以其为模板设计引物并引入EcoRⅠ和SalⅠ位点,PCR产物酶切后与pSANV2.0连接构建重组质粒pSANV2.0-DREAMshRNA-EGFP,酶切测序证实后,重组腺相关病毒介导将pSANV2.0-DREAMshRNA-EGFP感染PC12细胞,Western blot检测DREAM蛋白表达水平。结果经PCR、酶切及测序证实,重组腺相关病毒载体质粒pSANV2.0-DREAMshRNA-EGFP构建成功,包装成病毒感染PC12细胞48 h后,Western blot结果显示与对照组细胞相比,DREAM蛋白表达水平显著下降(P<0.05)。结论成功构建和筛选出介导特异性shRNA-DREAM的重组腺相关病毒载体。

Construction and Identification of DREAM Recombinant Adeno-Associated Virus Vector Carrying Small Interfering RNA

Objective To construct a DREAM recombinant adeno-associated virus vector carrying small interfering RNA(siRNA)and observe the inhibitory effects of DREAM protein expression in PC12 cells transfected with this vector.Methods We designed and synthesized the 64nt oligonucleotide containing the small hairpin of DREAM,which was inserted into the BamH Ⅰ and HindⅢ sites of pDC316-EGFP-U6 plasmid double digested by BamHⅠ and HindⅢ.Then we get resultant plasmid pDC316-EGFP-DREAMshRNA-U6,which served as template to designe primers.EcoRⅠ and SalⅠ sites were introduced into the primers,and PCR products after digestion with restrictive enzyme were ligated with pSANV2.0 to get resultant plasmid pSANV2.0-DREAMshRNA-EGFP.pSANV2.0-DREAMshRNA-EGFP was confirmed by sequencing and transfected into PC12 cells with adeno-associated virus.Western blot was used to detect the DREAM expression.Results The results of PCR,restrictive enzyme digestion and gene sequencing confirmed that the plasmid pSANV2.0-DREAMshRNA-EGFP had been constructed successfully.After it had been packed into adeno-associated virus to form rAAV2/1-DREAMshRNA-EGFP,it was used to transfect PC12 cells.Forty-eight h after transfection,the expression level of DREAM gene in PC12 cells transfected with rAAV2/1-DREAMshRNA-EGFP was descreased significantly as compared with the controls(P<0.05).Conclusion The recombinant adeno-associated virus vector with DREAM-targeted shRNAs is successfully constructed and screened.