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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) has the effect of promoting angiogenesis. It has been widely used in the treatment of bone defects, but its isoform VEGF121 is rarely studied. OBJECTIVE: To construct adenovirus expression vector carrying VEGF121-FLAG and hrGFP-1 gene and observe its expression in bone marrow stromal cells. Methods: VEGF121 gene was mutated by PCR using pTG19T-VEGF121 as a template to delete the stop codon of VEGF121 gene. Then, NotⅠand XhoⅠ restriction sites were added before and after the gene sequence, and the obtained gene was subcloned into pMD19-T plasmid The pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids were double-digested, and the small fragments and the large fragments were recovered from the gel, and the ligation reaction was carried out to construct the shuttle plasmid. BMSCs were infected after determination of recombinant virus titer and the fluorescence intensity was observed under a fluorescence microscope. RESULTS AND CONCLUSION: The recombinant adenovirus plasmid was successfully constructed by restriction enzyme digestion and DNA sequencing. The results of fluorescence microscopy showed that green fluorescent protein was expressed in bone marrow stromal cells infected with recombinant adenovirus. It can be seen that the constructed adenovirus expression vector carrying VEGF121-FLAG and hrGFP-1 genes can be expressed in eukaryotic cells and may be used for gene therapy of ischemic diseases.