甲基化转移酶Suv39h1基因慢病毒表达载体构建及鉴定  

Construction and Verification of Lentiviral Vector Targeting Suv39h1 Gene

作　　者：张帆[1] 卢韬[1] 孙荷[2] 吕娟[2] 杨明峰[2] 董学君[1,2] 

机构地区：[1]温州医学院检验医学院,生命科学学院,325035 [2]浙江省绍兴市人民医院,312000

出　　处：《医学研究杂志》2012年第12期47-50,共4页Journal of Medical Research

基　　金：浙江省自然科学基金资助项目(Y2090337)

摘　　要：目的构建小鼠H3K9甲基转移酶Suv39h1基因慢病表达毒载体。方法设计并合成带有Kpnl和Xmal酶切位点的引物,以携带Suv39h1 cDNA的PCMV-SPORT6载体为模板扩增目的基因,将其与Lenti-eGFP-Neo载体双酶切,T4连接酶连接,构建成PLenti-eGFP-Suv39h1重组载体,转化感受态细胞DH5α,PCR筛选阳性克隆质粒,酶切与测序鉴定正确后,将慢病毒四质粒系统共转染293T细胞,包装及效价测定。以感染复数(MOI值)为10和30的慢病毒颗粒感染293T细胞,RT-PCR检测Suv39h1 mRNA表达。结果 PCR、酶切及测序结果均显示目的片段插入正确,四质粒共转染293T细胞后,镜下可见95%的细胞表达绿色荧光;效价测定为2.11×108TU/ml;RT-PCR检测显示,MOI值为30的Suv39h1表达量是MOI值为10的3倍,两者均可在293T细胞中均匀稳定表达。结论成功构建Suv39h1基因慢病毒表达载体,为后续Suv39h1基因功能研究奠定了基础。Objective To construct the lentiviral vector recombined mouse H3K9 histone methyltransferase Suv39h1 gene. Methods PCR was performed to amplify the mouse Suv39h1 gene based on the PCMV - SPORT6 vector reeombined Suv39h1 , with the Kpnl and Xmal restriction sites primer. The product for amplification was cloned into lentiviral vector PLenti - eGFP - Neo by Kpnl and Xmal diges- tion and T4 ligase ligation. After transformating into competent E. coli cells,the candidate clones were identified by PCR and then were i- dentified according to the results of Suv39h1 PCR,recombined into PLenti- eGFP- Suv39h1 cleaved by restriction enzyme and DNA se- quences. The recombined vector was co -transfected into the 293T cells with four- plasmid system, package lentivirus particles,then and the viral titer was determined. Subsequently, the 293T cells were transfected by lentivirus with the values of multiplicity of infection （MOI） 10 and 30 separately,and the expression of Suv39h1 gene was detected by RT - PCR. Results Results of the PCR, restricting enzme digestion and DNA sequencing demonstrated that four -plasmid system successfully were transfected into 293T ceils in which 95% expressed enhanced green fluorescent protein （eGFP） ,and the functional titer of lentivirus particles was 2.11×108TU/ml. RT -PCR i- dentified Suv39h1 gene expression with MOI 30 as three times as MOI 10 which both could be stablely expressed in 293T cells. Conclu- sion Lentivirus expressing Suv39h1 was successfully constructed,which faciliatate further investigation of the roles Suv39h1 gene.

关 键 词：Suv39h1 组蛋白甲基转移酶 慢病毒载体 

分 类 号：R392.12[医药卫生—免疫学]