人肌细胞增强因子2C基因慢病毒载体的构建及其在293T细胞中的表达  

Construction of a lentiviral vector containing human MEF2C gene and its expression in 293T cells

作　　者：孙兆刚[1] 付鹏[1] 刘爱华[1] 王静波[1] 孙新六[1] 刘兴田[1] 

机构地区：[1]泰安市中心医院血液科,山东省271000

出　　处：《中华临床医师杂志（电子版）》2012年第20期80-82,共3页Chinese Journal of Clinicians(Electronic Edition)

基　　金：山东省自然科学基金(ZR2009CM132)

摘　　要：目的构建人肌细胞增强因子2C(MEF2C)慢病毒表达载体并检测其表达性能,以便应用过表达技术进一步研究MEF2C的功能。方法以人MEF2C基因序列为模板合成其编码框序列,通过限制性内切酶NotⅠ和NsiⅠ酶切、T4DNA连接酶连接,将MEF2C插入慢病毒载体质粒LV5-GFP,构建人MEF2C基因重组慢病毒质粒载体,转化感受态细菌,筛选阳性克隆,与包装质粒通过脂质体共转染人胚肾细胞系293T,进行慢病毒包装并测定病毒滴度。病毒感染293T细胞,荧光显微镜下观察感染效率,PCR方法检测MEF2C mRNA的过表达情况。结果成功构建慢病毒表达载体LV5-MEF2C-GFP,三质粒共转染293T细胞后形成假慢病毒颗粒;浓缩假病毒后测定其滴度为2×109TU/ml;以复感染系数MOI为5感染293T细胞,感染效率在70%以上。PCR证实感染带有目的基因假病毒的293T细胞表达MEF2CmRNA的水平较感染阴性对照病毒的293T细胞表达水平明显升高(t=11.93,P=0.000)。结论成功构建MEF2C慢病毒表达系统,为应用过表达技术探索其在肿瘤发生和发展中的作用奠定了基础。Objective To construct a lentiviral expressing vector carrying human MEF2C gene and assay its expression in 293T cells in order to further study its functions by overexpression technique.Methods The CDS region of MEF2C gene was synthetized according to the full-length MEF2C sequence from NCBI with PCR technology and then cloned into the lentiviral vector LV5-GFP by restriction endonucleases NotⅠand NsiⅠdigestion and T4 DNA ligase ligation.After transformation into competent E.coli cells,the candidate clones were identified by PCR and sequencing.The recombinant plasmid and the two packaging plasmids were co-transfected into human embryonic kidney cell line 293T cells by lipofectamine 2000 to produce the lentiviral particles,and the viral titer was determined.The 293T cells were infected by the lentiviral particles obtained and the transfection efficiency was assessed under fluorescent microscope.PCR technology was used to detect the overexpression level of MEF2C-mRNA in the transfected cells.Results The lentiviral vector LV5-MEF2C-GFP for MEF2C gene was successfully constructed.Strong green fluorescence was observed in 293T cells under fluorescent microscope after co-transfection of the cells with the 3 plasmids of lentiviral vector.The virus in the supernatant reached a titer of 2×109 TU/ml.The transfection efficiency of the collected virus exceeded 70% in 293T cells with a multiplicity of infection of 5.The cells infected with lentviral vector carring MEF2C gene had higher level of MEF2C-mRNA than the ones infected with lentivirus carring negative vector(t=11.93,P=0.000).Conclusions The lentiviral vector for human MEF2C has been successfully constructed with a high yield of lentivirus,which facilitate further investigation of the roles of MEF2C gene in the development and progression of cancer and other diseases.

关 键 词：慢病毒属 绿色荧光蛋白质类 MEF2C 293T细胞 

分 类 号：R730.2[医药卫生—肿瘤]