摘要
目的:制备含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体,为慢病毒载体的进一步广泛应用奠定基础。方法:克隆构建含分泌型萤光素酶和绿色荧光蛋白双报告基因的转基因载体pCS-gluc-2A-eGFP,酶切与序列分析鉴定其正确后,与包装质粒pCMVHR'Δ8.2、包膜质粒pVSV-G共转染293FT细胞,获得含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体;重组慢病毒载体感染A549、Huh7细胞后,用荧光显微镜直接观察报告基因GFP的表达,或取细胞上清实时检测分泌型萤光素酶的表达。结果:制备了含双报告基因的重组慢病毒载体,感染细胞后可以活体观察绿色荧光蛋白的表达,也可以快速灵敏地检测到分泌型萤光素酶的表达。结论:所获含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体感染效率高,表达易于活体实时检测,灵敏度高。本研究为慢病毒载体的广泛应用奠定了基础。
Objective：To prepare recombinant lentiviral vectors containing Gaussia luciferase and green fluorescent protein dual report gene,and lay the foundation for further wide application of lentiviral vector.Methods：We cloned and constructed the transfer plasmid containing dual report gene pCS-gluc-2A-eGFP,then packaging plasmid pCMVHR＇Δ8.2,envelope plasmid pVSV-G were cotransfected together with the above transfer plasmid into 293FT cells.48～72 hours later,the supernatant was collected and infected A549 and Huh7 to assess the expression of report gene.GFP reporter gene expression could be directly observed by fluorescence microscopy in cells and real-time detection of secreted luciferase（Gluc） expression was also available in supernatant.Results：We developed recombinant lentiviral vector containing dual report gene,the expression of GFP and secreted Gluc in infected cells could be detected rapidly and sensitively.Conclusion：The recombinant lentiviral vectors containing dual report gene were successfully developed with high infecting efficiency,the real-time expression of the GFP as well as the Gluc is easy to be live detected with high sensitivity.Our work paves ways for extensive application of lentiviral vectors.
出处
《生物技术通讯》
CAS
2011年第2期199-202,共4页
Letters in Biotechnology
基金
传染病重大专项(2009ZX1004-705
-715)
