多西环素调控的重组杆状病毒载体Ac-EGFP及Ac-HGF的构建  

Construction of recombinant baculovirus carriers of Ac-EGFPand Ac-HGFregulated by doxycycline

作　　者：潘志敏[1] 罗叶婷[2] 郭菲[3] 郑超[3] 马勇[3] 李广生[2] 程细高[1] 

机构地区：[1]南昌大学第二附属医院骨科,南昌330006 [2]赣州市人民医院神经内科,赣州341000 [3]南昌大学第一附属医院骨科,南昌330006

出　　处：《第二军医大学学报》2013年第8期904-908,共5页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81060147)~~

摘　　要：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 novel recombinant baculoviruses with Tet-On system and enhanced green fluorescent protein （EGFP） or hepatic growth factor （HGF） which could be regulated by different concentrations of doxycycline （DOX）. Methods The recombinant plasmids pFast-Tet, pTRE-EGFP and pTRE-HGF were digested. The target fragments were collected and connected to pFast-Tet, the resultants were used to transform DH10Bac competent cells containing AcMNPV Bacmid and helper plasmid, and the Bacmid DNA were identified （named Ac-EGFP and Ac-HGF） after selection and extraction. Ac-EGFP and Ac-HGF were then transfected into bone mesenchymal stem cells （BMSCs）, and the expression of EGFP and HGF were regulated by different concentrations of DOX （EGFP..0, 200, 500, and 1 000 ng/mL HGF 0, 10, 100, 500, 1 000, and 1 200 ng/mL） EGFP expression was observed under fluorescence microscope and the level of HGF expression was detected by ELISA. Results It was verified that Tet-On system was successfully constructed in a baculovirus vector with EGFP or HGF, and they were highly transfected into BMSCs. EGFP and HGF were highly expressed when exposed to high concentrations of DOX. And the expression of EGFP and HGF were gradually decreased at low concentration or absence of DOX. Conclusion Tet-On system can be used to construct a new recombinant baculovirus vector containing EGFP or HGF and it can stably and highly transfect BMSCs different concentrations of DOX can lead to different expression of EGFP and HGF, and they are in low background expression without DOX.

关 键 词：增强型绿色荧光蛋白 肝细胞生长因子 Tet—on系统 骨髓 间质干细胞 杆状病毒 

分 类 号：R394.8[医药卫生—医学遗传学]