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目的:构建携带人血管内皮生长因子(VEGF)基因的重组腺病毒载体,并转染体外培养的心肌细胞,检测VEGF的表达。万法:将人源性的VEGF165 cDNA正向插入到腺病毒载体PDC315,构建重组质粒,通过脂质体共转染293细胞,经同源重组获得携带人VEGF165基因的重组腺病毒,通过PCR扩增法鉴定所构建的腺病毒,扩增并测定滴度后,体外转染培养的心肌细胞,利用ELISA、Western印迹分析等方法检测VEGF在心肌细胞中的表达。结果:人VEGF165cDNA成功地正向插入到PDC315载体中,以重组病毒基因组DNA为模板,同时扩增出了610 bp的VEGF165cDNA基因片段,证实了所构建病毒的正确性,病毒滴度为2.8×108pfu／ml,Ad VEGF165体外转染心肌细胞3 d后,在培养细胞的上清液及细胞内检测到了VEGF的表达。结论:成功构建了表达人VEGF165基因的腺病毒载体,体外转染心肌细胞后能够满意表达VEGF,为基因治疗心肌缺血奠定基础。
Objective: To construct a recombinant adenovirus vector carrying human vascular endothelial growth factor (VEGF) gene and transfect the cultured cardiomyocytes to detect the expression of VEGF. Method: Human VEGF165 cDNA was inserted into the adenovirus vector PDC315 forward to construct a recombinant plasmid. The recombinant plasmid was co-transfected into 293 cells by liposome. Recombinant adenovirus carrying human VEGF165 gene was obtained by homologous recombination. The recombinant adenovirus was identified and amplified. The titer was determined and the cultured cardiomyocytes were transfected in vitro. The expression of VEGF in cardiomyocytes was detected by ELISA and Western blotting. Results: The human VEGF165 cDNA was successfully inserted into PDC315 vector successfully. The recombinant viral genomic DNA was used as a template and a 610 bp fragment of VEGF165 cDNA was amplified. The correctness of the constructed virus was confirmed. The virus titer was 2.8 × 108 pfu / Ml. Ad-VEGF165 transfected cardiomyocytes in vitro for 3 days, the expression of VEGF was detected in the supernatant and cells of cultured cells. CONCLUSION: The adenoviral vector expressing human VEGF165 gene has been successfully constructed. The expression of VEGF can be expressed satisfactorily after transfection of cardiomyocytes in vitro, which lays the foundation for gene therapy of myocardial ischemia.