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目的:拟构建人血管生成素1和血管内皮生长因子165腺病毒载体,观察靶细胞转染后目的基因的表达水平。方法:通过RT-PCR方法克隆人Ang-1全长编码基因,PCR法以pcDNA3.0-VEGF165质粒为模板扩增VEGF165基因,分别将目的基因酶切连接到带有GFP标记的pTrack-CMV质粒上,构建重组质粒pTrack-CMV-Ang-1和pTrack-CMV-VEGF165,经PCR、酶切和测序鉴定后将其PmeI线性化与腺病毒质粒pAdeasy-1共转化BJ5183细菌,获得重组腺病毒载体pAdeasy-1-pTrack-CMV-Ang-1/VEGF165,经PacI线性化后转染QBI-293A包装细胞,收获重组腺病毒Ad-Ang-1及Ad-VEGF165。结果:PCR﹑双酶切和测序鉴定结果证实成功构建pTrack-CMV-Ang-1/VEGF165重组转移质粒,PmeI线性化后重组腺病毒载体获得成功包装。脂质体转染QBI-293A细胞后光镜下可见由腺病毒引起的细胞圆缩、聚集呈菌落状的典型细胞病理性改变;荧光显微镜下可见绿色荧光蛋白的表达,且荧光强度随培养时间延长而逐渐增强;经多轮感染、扩增后,病毒效价可达(2.0～5.0)×1010pfu/mL。转染48h后,293A细胞中的血管生成素1与血管内皮生长因子含量均明显提高(F=427.93,17.93,P<0.05)。结论:成功构建并获得了Ad-Ang-1及Ad-VEGF165重组腺病毒,血管生成素1与血管内皮生长因子165均能在靶细胞中有效表达。
OBJECTIVE: To construct human adenoviral vector of human angiopoietin 1 and vascular endothelial growth factor 165 and to observe the expression level of the target gene after transfection. Methods: The full-length human Ang-1 encoding gene was cloned by RT-PCR and the VEGF165 gene was amplified by PCR using pcDNA3.0-VEGF165 as a template. The target gene was digested with GFP-tagged pTrack-CMV plasmid The recombinant plasmids pTrack-CMV-Ang-1 and pTrack-CMV-VEGF165 were constructed and identified by PCR, restriction endonuclease digestion and sequencing. The recombinant plasmids were linearized with PmeI and co-transformed into BJ5183 with the adenovirus plasmid pAdeasy-1 to obtain the recombinant adenoviral vector pAdeasy-1-pTrack-CMV-Ang-1 / VEGF165 was transfected into QBI-293A-packaged cells by PacI linearization. Recombinant adenovirus Ad-Ang-1 and Ad-VEGF165 were harvested. Results: The recombinant plasmid pTrack-CMV-Ang-1 / VEGF165 was successfully constructed by PCR, double enzyme digestion and sequencing. The PmeI linearized recombinant adenovirus vector was successfully packaged. After transfection of QBI-293A cells with lipofectamine 2000, the typical cell pathological changes caused by adenovirus were observed under light microscope. The expression of green fluorescent protein (GFP) was observed under fluorescence microscope. The fluorescence intensity of QBI- After several rounds of infection and amplification, the virus titer reached (2.0 ~ 5.0) × 1010pfu / mL. After 48h, the contents of angiopoietin 1 and VEGF in 293A cells were significantly increased (F = 427.93,17.93, P <0.05). Conclusion: Recombinant adenovirus Ad-Ang-1 and Ad-VEGF165 were successfully constructed and obtained. Both angiopoietin-1 and VEGF-165 could be efficiently expressed in target cells.