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目的构建携带报告基因绿色荧光蛋白(GFP)的重组腺病毒Ad5F11pTPEGFP,并且研究该病毒对人白血病细胞UT-7/Epo的感染效率及其在细胞中的复制。方法先通过分子克隆技术构建重组腺病毒质粒pAd5F11pTPEGFP,然后利用脂质体转染法,将其转染至HEK293细胞包装出重组腺病毒Ad5F11pTPEGFP,经酶切和PCR鉴定正确后,扩增并纯化得到滴度较高的重组腺病毒。以空载体病毒Ad5GFP作为对照,将纯化的重组腺病毒感染UT-7/Epo细胞,经流式细胞仪检测在不同感染复数(MOI)下荧光阳性细胞比例,即为Ad5F11pTPEGFP病毒对UT-7/Epo细胞的感染效率,同时将冻融后的重组腺病毒感染的UT-7/Epo细胞上清感染HEK293细胞,48 h后观察GFP在HEK293细胞中的表达。结果成功制备了重组腺病毒Ad5F11pTPEGFP,其对UT-7/Epo细胞的感染效率明显高于空载体对照病毒Ad5GFP,在MOI为200时,感染效率达98.2%,而空载体病毒在MOI为200时,对UT-7/Epo细胞的感染效率仅为29.7%。感染了重组腺病毒的UT-7/Epo细胞冻融上清感染新的HEK293细胞后,荧光显微镜下观察到GFP的表达。结论成功构建了重组腺病毒Ad5F11pTPEGFP,其对UT-7/Epo细胞有较高的感染效率,并能在其中复制。
Objective To construct a recombinant adenovirus Ad5F11pTPEGFP carrying reporter green fluorescent protein (GFP) and study its infection efficiency and its replication in human leukemia cells UT-7 / Epo. Methods The recombinant adenovirus plasmid pAd5F11pTPEGFP was constructed by molecular cloning technique and then transfected into HEK293 cells by lipofection. The recombinant adenovirus Ad5F11pTPEGFP was packaged and purified by restriction enzyme digestion and PCR. Titers of recombinant adenovirus. The Ad5GFP vector was used as a control to infect UT-7 / Epo cells with purified recombinant adenovirus. Flow cytometry was used to detect the percentage of fluorescent-positive cells at various MOIs. Epo cells. The HEK293 cells were infected with the supernatant of UT-7 / Epo cells infected with recombinant adenovirus after freezing and thawing. The expression of GFP in HEK293 cells was observed after 48 h. Results The recombinant adenovirus Ad5F11pTPEGFP was successfully prepared and the infection efficiency of UT-7 / Epo cells was significantly higher than that of empty control vector Ad5GFP. When the MOI was 200, the infection efficiency was 98.2%, while the empty vector was at MOI 200 , The infection rate of UT-7 / Epo cells was only 29.7%. After infection of recombinant adenovirus UT-7 / Epo cells freeze-thaw supernatant infected new HEK293 cells, the expression of GFP was observed under a fluorescence microscope. Conclusion The recombinant adenovirus Ad5F11pTPEGFP was constructed successfully, which has high infection efficiency and can replicate in UT-7 / Epo cells.