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

边素艳，盖鲁粤，叶平，杨月峰，王荣亮，王华，郭子宽，王立生

doi:10.3969/j.issn.1673-0364.2009.04.004

组织工程与重建外科杂志 >

2009 , Vol. 5 >Issue 2: 75 - 78

DOI: https://doi.org/10.3969/j.issn.1673-0364.2009.04.004

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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

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解放军总医院心血管二科;军事医学科学院放射医学研究所实验血液学研究室;

网络出版日期: 2020-07-23

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Construction of Recombinant Retroviral Vector Containing Firefly Luciferase Reporter Gene

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1 Second Department of Cardiology;Southern building;General Hospital of PLA;Beijing 100853;China.2 Department of Experimental hematology;Institute of Radiation Medicine;Academy of Military Medical Sciences;Beijing 100850;China;

Online published: 2020-07-23

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摘要

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。

关键词： 荧光素酶报告基因载体; pGL3-Basic; 重组逆转录病毒载体; pLXSN; 构建

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边素艳，盖鲁粤，叶平，杨月峰，王荣亮，王华，郭子宽，王立生 . 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[J]. 组织工程与重建外科杂志, 2009 , 5(2) : 75 -78 . DOI: 10.3969/j.issn.1673-0364.2009.04.004

Abstract

Objective To construct recombinant retroviral vector carrying firefly 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 res...

Key words： Firefly luciferase reporter gene（fluc） vector; pGL3-Basic; Recombinant retroviral vector; pLXSN; Construction

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