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目的构建携带人血管内皮细胞生长因子121(HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR121,HVEGF121)和增强型绿色荧光蛋白(ENHANCED GREEN FLUORESCENT PROTEIN,EGFP)融合基因的腺病毒表达载体,观测其表达。方法以PUC18-HVEGF121质粒中的HVEGF121CDNA为模板,用PCR法扩增HVEGF121CDNA片段并去除其终止密码子,将此片段连接到含有EGFP CDNA的PEGFP-N1质粒中,再将融合基因HVEGF121-EGFP克隆到穿梭质粒PDC315中,与腺病毒质粒共同转染293细胞,构建重组腺病毒,并进行滴度测定。用重组腺病毒感染NIH3T3细胞,在荧光显微镜下观察荧光强度,并用免疫组化法检测VEGF121在NIH3T3中的表达。结果经酶切鉴定及基因测序证实重组腺病毒质粒构建成功,病毒滴度为1.2×1010~2.7×1011PFU/ML。荧光显微镜下观察表明,感染重组腺病毒的NIH3T3细胞有明显的绿色荧光表达。免疫组化结果也证明VEGF121能在NIH3T3中的表达。结论构建的HVEGF121-EGFP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,并观察外源基因在体内的表达。
Objective To construct adenovirus expression vector carrying human VASCULAR ENDOTHELIAL GROWTH FACTOR121 (HVEGF121) and ENHANCED GREEN FLUORESCENT PROTEIN (EGFP) fusion gene and observe their expression. Methods HVEGF121 cDNA of PUC18-HVEGF121 plasmid was used as a template to amplify the HVEGF121 cDNA fragment by PCR and remove its stop codon. The fragment was ligated into PEGFP-N1 plasmid containing EGFP CDNA, and the fusion gene HVEGF121-EGFP was cloned into Shuttle plasmid PDC315, co-transfected with adenovirus plasmid 293 cells, the construction of recombinant adenovirus, and titer determination. NIH3T3 cells were infected with recombinant adenovirus, the fluorescence intensity was observed under a fluorescence microscope, and the expression of VEGF121 in NIH3T3 was detected by immunohistochemistry. Results Recombinant adenovirus plasmid was successfully constructed by restriction enzyme digestion and sequencing. The virus titer was 1.2 × 1010 ~ 2.7 × 1011 PFU / ML. Fluorescence microscopy showed that NIH3T3 cells infected with recombinant adenovirus had obvious green fluorescence. Immunohistochemical results also demonstrated VEGF121 expression in NIH3T3. CONCLUSION: The constructed HVEGF121-EGFP adenovirus expression vector can be expressed in eukaryotic cells and may be used in gene therapy of ischemic diseases and to observe the expression of foreign genes in vivo.