杆状病毒介导LacZ基因转染体外培养的人虹膜色素上皮细胞

摘要

Objective:To evaluate the feasibility of recombinant baculovirus mediated transduction of LacZ reporter gene to cultured human iris pigment epithelial(IPE) cells in vitro.Methods:Transfer vector pBac-β was constructed. The recombinant baculovirus BV-β was produced using the Bac-to-Bac system. Human IPE cells were cultured and identified by immunocytochemical staining. Then IPE cells were transduced by recombinant virus BV-β inoculum at MOIs of 200. After IPE cells were transducted for 24 hr,observed for β-Gal activity on an inverted microscope. Blue X-gal-positive cells were counted and the transduction efficiencies were expressed as the percentage of positive cells of the total number of cells.Results:Successful construction of transfer vector pBac-β and recombinant baculovirus BV-β were confirmed by restriction analysis and sequencing. Cultured IPE showed cytokeratin and S2100 protein positive under immunochemistry study. β-Galactosidase analysis showed that 85% IPE cells are stained blue in 24 hours after infection.Conclusion:The cultured human IPE cells seemed to be susceptible to baculovirus transduction in vitro. The baculovirus vectors were demonstrated to have the potential capability as a tool to generate genetically modified autologous IPE cells expressing biological therapeutics.%目的:研究重组杆状病毒(baculovirus)载体介导β-半乳糖苷酶基因(LacZ)对体外培养虹膜色素上皮(IPE)细胞的转染和表达情况.方法:将质粒pCMV-β中的CMV-LacZ表达盒插入杆状病毒表达载体pFast BacI的Eco RI/Hind Ⅲ位点,构建重组质粒pBac-β,并利用Bac-to-Bac表达系统在Sf9细胞中产生出重组杆状病毒BV-β.将MOI为200的BV-β感染经酶消化加显微法分离培养的IPE细胞,24 h后进行X-gal染色.在显微镜下观察IPE中LacZ表达阳性的情况,记录阳性细胞所占的百分比.结果:限制性酶切分析及测序结果表明,我们已成功构建表达质粒pBac-β及重组杆状病毒BV-β.X-gal染色证实杆状病毒介导LacZ在细胞中的表达呈蓝色,弥漫于整个胞浆.感染后24 h时表达阳性率达85%.结论:重组杆状病毒可有效介导报告基因在体外培养的IPE细胞中表达,为利用杆状病毒构建可供移植的基因工程化虹膜色素上皮细胞提供了实验依据.