大鼠VEGF腺病毒基因转染系统的构建及鉴定

目的:探讨构建携带大鼠血管内皮细胞生长因子(VEGF)的重组腺病毒载体方法，为后续的基因转染研究作准备。方法:采用RT-PCR扩增的方法获取鼠源性的VEGF,并克隆入穿梭质粒pDC316。构建的质粒pDC316-VEGF经酶切及测序鉴定正确后，通过lipofectamine2000的介导与腺病毒包装质粒pBHGE3共转染至人胚肾细胞HEK293，经同源重组后获得携带鼠VEGF的重组腺病毒VDC316-VEGF。应用PCR鉴定重组腺病毒，空斑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:PCR鉴定证实重组腺病毒含有鼠VEGF,病毒滴度为3×109pfu/mL。结论:成功构建的携带大鼠VEGF的重组腺病毒载体能在HEK293细胞内扩增获得足够高的病毒滴度,可作为后续基因治疗研究工作中可靠的基因转染工具。

Construction and identification of rat vascular endothelial growth factor recombinant adenovirus-mediated gene transfer system

Abstract:Objective:To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor (VEGF) as preparation for later use for genetic transfection.Methods :Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF, VDC316-VEGF, was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose (TCID50) assay.Results:The newly constructed recombinant adenovirus was confirmed carrying rat VEGF by PCR, and its titration value determined based on TCID50 assay was 3×109pfu／ml.Conclusions:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.