Eμ-VH启动子调控c-Myc表达慢病毒载体的构建及其体外功能  

Construction and function of a lentiviral vector harboring c-Myc gene under the control of Eμ-VH promoter

作　　者：陈琳[1] 陈燕[1] 肖东[1] 

机构地区：[1]南方医科大学,广州510515

出　　处：《山东医药》2016年第21期1-3,共3页Shandong Medical Journal

基　　金：广东省产业技术研究与开发专项资金资助项目(2013B060300013)

摘　　要：目的构建人B淋巴细胞特异性启动子Eμ-VH调控癌基因c-Myc表达的慢病毒载体(pEMIR),并探讨其体外功能。方法以pLV-c-Myc-IRES-增强绿色荧光蛋白(EGFP)为模板,经PCR扩增、酶切后得到pEVCHR;以pEVCHR为模板,PCR扩增、酶切后得到慢病毒载体pEMIR,并行测序和酶切鉴定。将pEMIR分别瞬时转染入人源胚胎肾细胞293T、人弥漫大B细胞淋巴瘤细胞株(OCL-Ly3、OCL-Ly10),倒置荧光显微镜检测各细胞EGFP和单体红色荧光蛋白(mRFP)的表达,用qRT-PCR法检测细胞内c-Myc mRNA表达,用Western blotting法检测293T细胞c-Myc蛋白表达。结果 Eμ-VH-c-Myc-H2BmRFP基因片段电泳结果与预测值4 129 bp相符,Vector DNA电泳可见一条带与预测值8 561 bp相符;pEMIR酶切并电泳的结果与理论预测值相符;对pEMIR质粒进行测序,结果与预期相符,证明pEMIR构建成功。三种细胞经pEMIR转染后,倒置荧光显微镜下可见绿色和红色荧光,同时转基因c-Myc表达正常。结论成功构建Eμ-VH启动子调控c-Myc表达的慢病毒载体,体外实验证明该载体能够在人正常细胞和肿瘤细胞中均可发挥功能。Objective To construct a lentiviral vector harboring c-Myc gene under the control of human B lymphocytespecific Eμ-VH promoter（ p EMIR）,and to investigate its function. Methods Firstly,taking p LV-c-Myc-IRES-EGFP as a template,after PCR amplification and enzyme digestion,we got p EVCHR. Secondly,taking plasmid p EVCHR as a template,after PCR amplification and enzyme digestion,we got a lentiviral vector p EMIR. p EMIR was transiently transfected into human embryonic kidney cells 293 T and human diffuse large B-cell lymphoma cell line（ OCL-Ly3 or OCL-Ly10）. The expression of enhanced green fluorescent protein（ EGFP） and mono-red fluorescence protein（ mRFP） was detected under inverted fluorescence microscope,and the expression of c-Myc mRNA and protein was detected by qRT-PCR and Western blotting. Results The electrophoresis results of Eμ--VH-c-Myc-H2 BmRFP gene fragment were in agreement with the predicted value of 4129 bp,and the electrophoresis of the vector DNA showed a band that was consistent with the predicted value of 8561 bp. Enzyme digestion and DNA sequencing demonstrated that p EMIR was successfully constructed. Cells transiently transfected with p EMIR displayed the green and red fluorescence under inverted fluorescence microscope,and the expression of c-Myc was normal. Conclusion The lentiviral vector harboring c-Myc gene under the control of Eμ-VH promoter is successfully constructed,and the experiments in vitro demonstrate that it can be effective in human normal cells and tumor cells.

关 键 词：Eμ-VH启动子 C-MYC癌基因 慢病毒载体 弥漫大B细胞淋巴瘤 

分 类 号：R394[医药卫生—医学遗传学]