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目的:构建人粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因与EB病毒基因LMP2A融合基因GC2A的重组腺病毒表达载体。方法:将构建的融合基因GC2A定向亚克隆至质粒pAdTrack-CMV上,经酶切及电泳检测后,PmeL酶切pAdTrack-CMV-GC2A,与质粒pAdEasy-1一起电转化E.coli.BJ5183,筛选重组菌,经PacⅠ酶切鉴定后用磷酸钙法转染QBI-293A细胞,包装成重组腺病毒vAd-GC2A,采用pAdTrack-CMV-GC2A中报告基因GFP的表达,观测其表达情况。结果:采用内切酶Bg1Ⅱ和EcoRⅤ对质粒pAdTrack-CMV-GC2A进行双酶切分析,电泳后可观察到约1 961和9 200bp的片段。重组腺病毒载体经PacⅠ酶切后观察到约33kb的DNA片段和4.5kb的DNA片段,说明pAdTrack-CMV-GC2A和pAdEasy-1的重组发生在ori和右臂区,pAd-GC2A经PacⅠ酶切线性化后,磷酸钙法转染293细胞,56h后在荧光倒置显微镜下可看到有绿色荧光出现,说明已转染成功,并能在293细胞中表达。结论:融合基因GC2A正确插入,真核表达载体vAd-GC2A成功构建,在293细胞中包装获得重组腺病毒。
OBJECTIVE: To construct a recombinant adenovirus expressing human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and Epstein-Barr virus (LMP2A) fusion gene GC2A. Methods: The constructed fusion gene GC2A was subcloned into plasmid pAdTrack-CMV. After digestion and electrophoresis, PmeL was digested with pAdTrack-CMV-GC2A and electroporated into E.coli.BJ5183 with plasmid pAdEasy-1 to screen The recombinants were digested with Pac Ⅰ and then transfected into QBI-293A cells by calcium phosphate method. The recombinant plasmids were packaged into recombinant adenovirus vAd-GC2A and the expression of GFP was detected by pAdTrack-CMV-GC2A. Results: The plasmid pAdTrack-CMV-GC2A was digested with endonucleases Bg1Ⅱ and EcoRⅤ. After electrophoresis, about 1 961 and 9 200 bp fragments were observed. The recombinant adenovirus vector was digested with Pac Ⅰ and the DNA fragment of about 33 kb and the DNA fragment of 4.5 kb were observed. The recombination of pAdTrack-CMV-GC2A and pAdEasy-1 occurred in ori and the right arm region. The pAd-GC2A was digested with PacⅠ After linearization, 293 cells were transfected with calcium phosphate. After 56 hours, green fluorescence appeared under the fluorescence inverted microscope, indicating that the transfection was successful and could be expressed in 293 cells. Conclusion: The fusion gene GC2A was inserted correctly and the eukaryotic expression vector vAd-GC2A was successfully constructed. The recombinant adenovirus was packaged in 293 cells.