重组腺病毒载体Ad5-Olig1-eGFP的构建与鉴定

Construction and identification of recombinant adenovirus vector Ad5-Olig1-eGFP

目的:转录因子Olig1调控了少突胶质前体细胞的分化和成熟,并参与了脱髓鞘损伤后的修复,是治疗脱髓鞘疾病的候选基因,在缺血性脑血管病的治疗中具有极大的应用价值。本实验旨在构建携带Olig1基因的重组腺病毒载体。方法:pDC315-Olig1为模板,聚合酶链反应扩增酶切Olig1片段,连接到带有eGFP标记基因的载体质粒pDC315-eGFP上,构建穿梭质粒pDC315-Olig1-eGFP,经PCR,酶切及测序鉴定后,利用AdMax包装系统,通过脂质体2000的介导与骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞,同源重组后产生复制缺陷型重组腺病毒载体Ad5-Olig1-eGFP,应用PCR对毒种进行鉴定,传代纯化病毒并反复冻融扩增病毒。以50%组织培养感染剂量法(TCID50)测定病毒滴度。结果:经聚合酶链反应,酶切鉴定和测序分析,得到大小700bp(Olig1)目的片段,证实正确构建重组穿梭质粒pDC315-Olig1-eGFP和重组腺病毒Ad5-Olig1-eGFP,测得重组腺病毒颗粒数为2.3×1011v.p./mL。结论:成功构建携带Olig1的重组腺病毒载体Ad5-Olig1-eGFP,为缺血性脑血管病的基因治疗提供基因载体并奠定实验基础。

Objective Transcription factor Olig1 plays an important role in oligodendrocytes differentiation and maturation.To construct the recombinant adenovirus vector carrying Olig1 gene.Methods The Olig1 gene was amplified by PCR from plasmid pDC315-Olig1.The Olig1 gene was inserted into pDC315-eGFP vector to construct shuttle plasmid pDC315-Olig1-eGFP.After confirmation by PCR and sequencing analysis,pDC315-Olig1-eGFP was cotransfected with the adenovirus skeleton plasmid pBHGlox-E1.3Cre into HEK 293 cells to produce replication defective recombinant adenovirus Ad5-Olig1-eGFP.Ad5-Olig1-eGFP was subsequently identified using PCR.The recombinant adenovirus was propagated by repeat infection of HEK 293 cells,and the virus titer was determined by 50% tissue culture dose（TCDI50） assay.Results PCR amplification,restriction digestion and sequencing analysis was performed to identify both the recombinant shuttle plasmid pDC316-Olig1-eGFP and the recombinant adenovirus vector Ad5-Olig1-eGFP were correctly constructed.The titer of recombinant adenovirus was 2.3 ×1011 v.p./mL.Conclusion Recombinant adenovirus Ad5-Olig1-eGFP was successfully constructed,which laid a foundation for future ischemic cerebral therapy study.