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目的将Tet-On系统与增强型绿色荧光蛋白(EGFP)或肝细胞生长因子(HGF)共同构建于一新型重组杆状病毒载体,并以不同浓度的多西环素(DOX)调控EGFP及HGF表达。方法酶切重组质粒pFast-Tet、pTRE-EGFP和pTRE-HGF,回收目的片段后连接pFast-Tet,分别转化含有AcMNPV Bacmid和helper质粒的DH10Bac感受态细胞,筛选后提取Bacmid DNA并鉴定(命名为Ac-EGFP和Ac-HGF)。将Ac-EGFP和Ac-HGF转染骨髓间充质干细胞,加入不同浓度的DOX调控EGFP(DOX浓度为0、200、500、1000ng/mL)和HGF(DOX浓度为0、10、100、500、1000、1200ng/mL)的表达。在荧光显微镜下观察EGFP的表达,用ELISA法检测HGF的表达量。结果经鉴定EGFP、HGF与Tet-On系统成功构建在同一杆状病毒载体,且在骨髓间充质干细胞中具有较高的转染率。在较高浓度DOX调控下改造后的重组杆状病毒可高表达EGFP和HGF,低浓度或无DOX时表达量逐渐减低。结论本研究证实可将Tet-On系统与EGFP或HGF共同构建于一新型重组杆状病毒载体,改造后的重组杆状病毒能稳定、高效转染骨髓间充质干细胞,不同浓度的DOX可调控EGFP及HGF的表达,无DOX时呈低本底。
Objective To construct a novel recombinant baculovirus vector with Tet-On system and enhanced green fluorescent protein (EGFP) or hepatocyte growth factor (HGF) and to regulate EGFP and HGF with different concentrations of doxorubicin (DOX) expression. Methods Recombinant plasmids pFast-Tet, pTRE-EGFP and pTRE-HGF were digested by restriction endonucleases. After the target fragment was recovered and ligated into pFast-Tet, DH10Bac competent cells containing AcMNPV Bacmid and helper plasmids were transformed respectively. Bacmid DNA was extracted and identified Ac-EGFP and Ac-HGF). Bone marrow mesenchymal stem cells were transfected with Ac-EGFP and Ac-HGF, and different concentrations of DOX were added to control EGFP (DOX concentration: 0,200,500,1000ng / mL) and HGF (DOX concentration: 0,10,100,500 , 1000, 1200 ng / mL). The expression of EGFP was observed under a fluorescence microscope, and the expression of HGF was detected by ELISA. Results The identified EGFP, HGF and Tet-On systems were successfully constructed on the same baculovirus vector and had higher transfection efficiency in bone marrow mesenchymal stem cells. The recombinant baculovirus transformed with higher concentration of DOX could up-regulate the expression of EGFP and HGF, while the expression of EGFP and HGF decreased gradually with low concentration or without DOX. Conclusion This study confirmed that the Tet-On system can be constructed together with EGFP or HGF in a novel recombinant baculovirus vector. The recombinant baculovirus can stably and efficiently transfect bone marrow mesenchymal stem cells. Different concentrations of DOX can be regulated EGFP and HGF expression, low DOX-free background.