萤火虫荧光素酶报告基因重组逆转录病毒载体的构建及鉴定

目的构建含萤火虫荧光素酶报告基因的逆转录病毒载体,为进一步研究生物发光活体动物体内光学成像奠定基础。方法利用重组DNA技术,将荧光素酶报告基因载体pGL3-Basic双酶切得到的目的基因fluc+亚克隆至逆转录病毒载体pLXSN中,重组质粒pL(fluc)SN在脂质体介导下乒乓转染GP+E86和PA317包装细胞,G418筛选,直至出现抗性克隆。扩大培养,测定病毒滴度,并检测荧光素酶活性。结果经限制性酶切分析鉴定,载体插入基因大小、位置均正确,并用GP+E86和PA317细胞进行包装、病毒滴度测定、筛选,建立具有较高滴度的重组逆转录病毒fluc细胞系,该细胞可高效表达荧光素酶。结论成功构建了重组质粒pL(fluc)SN,为标识干细胞进行基础研究提供了一种检测方法和平台。

Construction of Recombinant Retroviral Vector Containing Firefly Luciferase Reporter Gene

Objective To construct recombinant retroviral vector carrying firefy luciferase reporter gene （fluc） for further study of bioluminescence imaging in vivo. Methods Target gene-fluc was obtained by cutting firefly luciferase reporter gene （fluc） vector pGL3-Basic using restriction enzyme digestion. Then fluc gene was sub-cloned into retroviral vector pLXSN. GP＋E86 and PA317 pakage cells were infected by the obtained recombinant retroviral vector pL （fluc）SN with ping-pong infection assay and screened with G418 resistance. The stable expression of the fluc gene in positive clones was identified by detecting luciferase enzyme activity. Results Enzyme digestion indicated that the retroviral vector pL（fuc）SN was successfully constructed. The fluc gene was integrated into the PA317 and GP＋E86 genome and expressed stably in the host cells. Conclusion The target retroviral vector pL （fluc） SN was constructed successfully. It provides an effective tool or platform for basic research of tracing grafted cells.