【摘要】 目的利用串联表达质粒pEGFP6-1-siFas1+siFas2和BDAdeno-X系统构建腺病毒表达Fas-shRNA载体。方法双酶切pEGFP6-1-siFas1+siFas2串联表达质粒和pShuttle2质粒,T4DNA连接酶连接胶回收片段,转化感受态Escherichiacoli(E.coli)DH5α菌株,挑取单克隆菌落LB(Luria-Bertani)培养基中扩增培养;小量质粒试剂盒提取pShuttle2-siFas1+siFas2质粒,酶切鉴定;双酶切pShuttle2-siFas1+siFas2质粒,T4DNA连接酶连接酶切产物和腺病毒BDAdeno-XViralDNA。回收连接产物,SwaI酶切去除未重组的腺病毒质粒,再回收酶切产物,转化感受态E.coliDH5α;聚合酶链反应(PCR)筛选鉴定阳性克隆;提取pAdeno-siFas1+siFas2质粒,进一步酶切鉴定;重组腺病毒pAdeno-siFas1+siFas2质粒线性化,经脂质体转染HEK293A细胞;收细胞进行裂解收病毒。结果构建表达2个Fas-shRNA的串联重组腺病毒载体pAdeno-siFas1+siFas2,经酶切鉴定和PCR分析,重组腺病毒质粒构建正确;经HEK293A细胞包装成功。结论成功构建表达2个Fas-shRNA的串联重组腺病毒载体pAdeno-siFas1+siFas2,为进一步转染细胞和感染小鼠抑制Fas基因表达奠定了基础。更多还原

【Abstract】 Objective To construct the recombinant adenovirus vector expressing two Fas-targeted short hairpin RNA(shRNA)by pEGFP6-1-siFas1+siFas2 and BD Adeno-X DNA.Methods The pEGFP6-1-siFas1+siFas2 and pShuttle2 were digested by restriction endonuclease.A long segment from pShuttle2 and a short segment from pEGFP6-1-siFas1+siFas2 were reclaimed,and connected with T4 DNA ligase.Then the obtained recombinant plasmids were transformed into the Escherichia coli strain DH5α for screening and amplifying.The plasmids were identified by restriction enzyme and PCR.The plasmid pShuttle2-siFas1+siFas2 was digested to completion with PI-Sce I and I-Ceu I.A 2706bp segment from pShuttle2-siFas1+siFas2 and BD Adeno-X Viral DNA were connected with T4 DNA ligase.The recombinant plasmid series were transformed into the Escherichia coli strain DH5α for amplifying.The recombinant plasmid series was identified by PI-SceⅠ+I-CeuⅠcleavage.pAdeno-siFas1+siFas2 was digested by PacⅠcompletely.Linear pAdeno-siFas1+siFas2 was transformed into HEK293 cell with METAFECTENE(tm).The adenovirus vector was packed,and then the adenovirus was reclaimed based on the presence of cytopathic effect.Results The adenovirus vector with two Fas-targeted shRNA expressing frames were successfully constructed and packed correctly in HEK293 cells.Conclusion Successful construction of the recombinant adenovirus vector with two Fas-targeted shRNAs provided a basis for further studies on cell transfection and suppression of Fas in infected mice.更多还原