构建携带VEGF_(121)-FLAG和hrGFP-1基因腺病毒表达载体在骨髓基质干细胞中的表达(英文)

Construction of adenovirus vectors carrying VEGF_(121)-FLAG and hrGFP-1 and their expressions in bone marrow stromal stem cells

背景:血管内皮生长因子具有促进血管生成的作用,在骨缺损的治疗研究中运用较多,但其异构体VEGF121的研究较少。目的:构建携带VEGF121-FLAG和hrGFP-1基因的腺病毒表达载体并观测其在骨髓基质干细胞中的表达。方法:以pTG19T-VEGF121为模板利用PCR技术突变VEGF121基因以去除VEGF121基因中终止密码子,之后在基因序列前后分别加入NotⅠ和XhoⅠ酶切位点,并将得到的基因亚克隆至pMD19-T质粒上,双酶切pMD19-T-VEGF121和pShuttle-CMV-IRES-hrGFP-1质粒,凝胶回收小片段和大片段,后进行连接反应,完成穿梭质粒的构建。重组病毒物理滴度测定后感染骨髓基质干细胞,在荧光显微镜下观察荧光强度。结果与结论:经酶切鉴定及基因测序证实重组腺病毒质粒构建成功,荧光显微镜下观察表明,感染重组腺病毒的骨髓基质干细胞有明显的绿色荧光表达。可见构建的携带VEGF121-FLAG和hrGFP-1基因的腺病毒表达载体可在真核细胞表达,有可能用于缺血性疾患的基因治疗。

BACKGROUND：Vascular endothelial growth factor（VEGF） can promote angiogenesis,and has been extensively used in treatment of bone defect.However,few studies have addressed its isomer VEGF121.OBJECTIVE：To construct adenovirus vector carrying VEGF121-FLAG and humanized Renilla reniformis green fluorescent protein 1（hrGFP-1） and observe its expression in bone marrow stromal stem cells（BMSCs）.METHODS：Using polymerase chain reaction technique,VEGF121 gene contained in the plasmid of pTG19T-VEGF121 was used to remove termination codon.NotI and Xho I restriction sites were added before and after gene sequence.Obtained gene subclone was moved onto pMD19-T plasmid.The pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids underwent double enzymatic digestion.Small fragment and big fragment were retrieved utilizing gel.Subsequently,coupled reaction was conducted to complete the construction of shuttle plasmid.After measuring virus titer,BMSCs were transfected and the fluorescence intensity was observed under fluorescence microscope.RESULTS AND CONCLUSION：Recombinant adenovirus plasmid was successfully constructed by enzymatic digestion determination and gene sequence.Fluorescence microscope has shown that BMSCs transfected with recombinant adenovirus presented significantly green fluorescence expression.Thus,adenovirus vector carrying VEGF121-FLAG and hrGFP-1 gene can express in eukaryotic cells,which can be used for gene therapy for ischemic disease.