膜锚定Gaussia萤光素酶的细胞标记及生物荧光成像  

Bioluminescence Imaging Cells Labeled with Membrane-Anchored Form of Gaussia Luciferase

作　　者：樊炜[1] 贾帅争[1] 王怡[1] 阎少多[1] 高博[1] 彭剑淳[1] 詹林盛[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《生物技术通讯》2011年第6期838-841,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30972615;81072497);北京市自然科学基金(7112104)

摘　　要：目的:制备表达膜锚定Gaussia萤光素酶(extGluc)报告基因的慢病毒,用于标记细胞。方法:将报告基因extGluc克隆至慢病毒载体pCCsin.PPT.SFFV.IRES.eGFP.Wpre(VeGFP)中,以聚乙烯亚胺(PEI)介导,将慢病毒包装所需4种质粒(pVeGFP-extGLuc、pMDL、pRev、pVSVG),转染293FT细胞,72 h后收集病毒上清进行浓缩,感染293FT细胞,并用流式细胞仪检测病毒滴度,生物荧光成像和化学发光分析extGluc的表达;之后,用收集的慢病毒感染人单核细胞白血病细胞株U937。结果:对经PCR筛选出的阳性克隆所含质粒进行酶切鉴定,表明extGlu报告基因插入载体中;重组慢病毒包装成功且病毒滴度为5×106 TU/mL;用包装的病毒颗粒感染293FT细胞,生物荧光成像和化学发光证实extGluc的膜定位,且酶活性与细胞数目呈线性相关;病毒颗粒能够感染悬浮细胞U937。结论:包装了extGluc标记的重组慢病毒,可用于标记细胞,为体内监测细胞迁移、聚集和变化提供了一种方法。Objective： To produce lentivirus expressing reporter gene membrane-anchored form of Gaussia luciferase（extGluc） used for bioluminescence imaging cells.Methods： Reporter gene extGluc was cloned into pCCsin.PPT.SFFV.IRES.eGFP.Wpre（VeGFP）.pVeGFP-extGLuc,pMDL,pRev and pVSVG,which were required for packaging,were cotransfected into 293FT cells mediated by polyethylenimine branched.Lentivirus was collected at 72 h post-transfection,concentrated and then was used to infect 293FT cells.Viral titer was determined by flow cytometry.Luciferase activity was detected by bioluminescence imaging and chemiluminescence.At last,human monocytic leukemia cell line U937 were infected by viral supernatant.Results： PCR and enzyme digestion results indicated that reporter gene extGLuc was successfully cloned into VeGFP.The lentivirus was packaged successfully.The lentivirus titer was 5×106 TU/mL.After infecting 293FT with virus particles,extGluc was confirmed to be membrane-anchored by using bioluminescence imaging and chemiluminescence.And the luciferase activity was linear correlated with cell numbers.The virus could infect U937 cell line.Conclusion： The recombinant lentivirus labeled with extGluc was packaged successfully.It will provide an effective tool for detecting cells in vivo.

关 键 词：Gaussia萤光素酶 膜锚定 慢病毒包装 生物荧光成像 

分 类 号：Q2[生物学—细胞生物学] Q78