人血管生成素1和血管内皮生长因子165重组腺病毒载体构建及目的基因表达

Construction and target gene expression of adenovirus vectors containing angiogenin-1/vascular endothelial growth factor 165

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。

METHODS： The pTrack-CMV- Ang-1/VEGF165 was constructed by RT-PCR with cloned sequence of Ang-1 and PCR using pcDNA3.0-VEGF165 recombined plasmid as template, enzyme digestion and ligation. The pTrack-CMV-Ang-1/VEGF165 lineared by PmeI was co-transformed into B J5183 with pAdeasy-1. The pAdeasy-1-pTrack-CMV-Ang-1/VEGF165 recombined adenovirus vectors were lineared with Pacl and then transfected into QBI-293A cells. The Ang-1/VEGF165 recombined adenovirus was obtained. RESULTS： PCR, double digestion and DNA sequencing confirmed that, the pTrack-CMV-Ang-1/VEGF165 recombinant transfer plasmid could be successfully constructed, the recombined adenovirus vector were successfully packaged with the titer being as high as （2.0-5.0）×10^10 pfu/mL. After QBI-293A was transfected with liposome, typical pathological changes induced by adenovirus, such as cell volume shrinkage and aggregation as colony, were noted under light microscope. Fluorescence microscope showed the expression of green fluorescent protein, and fluorescence intensity enhanced with the time going. At 28 hours of transfection, the contents of Ang-1 and VEGF165 in 293A cells were obviously increased （F=427.93, 17.93, P 〈 0.05）. CONCLUSION： The recombined adenovirus （Ad-Ang-INEGF165） can be constructed successfully. Both Ang-1 and VEGF165 effectively express in target cells.