小鼠甲基转移酶EZH2基因慢病毒表达载体的构建及验证

Construction and Verification of Mouse Histone Methyltransferase EZH2 Gene Lentiviral Vector

目的构建小鼠组蛋白H3K27三甲基转移酶EZH2基因慢病毒载体及鉴定。方法以携带EZH2 cDNA的PCMV-SPORT6载体为模板,自行设计携带有Kpnl和Xmal酶切位点的引物PCR扩增出目的基因编码序列,扩增产物用内切酶酶切后定向克隆到慢病毒载体PLenti-eGFP-NEO中,通过PCR、酶切及测序验证载体;将重组慢病毒载体和包装质粒pRsv-REV、pMDlg-pRRE及pMD2G组成的四质粒系统,共转染293T细胞包装成慢病毒,收集含病毒颗粒的细胞上清液,浓缩和纯化后得到高效价的病毒液,转染293T细胞进行效价测定。结果 PCR扩增出约2241bp的序列,构建的慢病毒载体测序结果与Gene-bank报道的目的基因序列一致;四质粒系统成功共转染入293T细胞中,在细胞中能够稳定表达,包装出了2.1×108TU/ml的病毒液。结论成功构建了小鼠EZH2基因慢病毒载体,并得到2.1×108TU/ml高效价的病毒液。

Objective To construct and identify of the Lentiviral vector recombined mouse histone methyhransferase EZH2 gene. Methods PCR was performed to amplify the mouse EZH2 gene based on the PCMV - SPORT6 vector recombined EZH2,with the Kpnl and Xmal restriction sites primer. The product for amplification was cloned into Lentiviral vector PLenti - eGFP - NEO after being cleaved by Kpnl and Xmal,and then was identified according to the results of EZH2 PCR, recombined PLenti - eGFP - EZH2 cleaved by restriction enzyme and DNA sequences. The recombined vector was co - transfected into the 293T cells with four - plasmid system, pRsv - REV, pMDlg- pRRE and pMD2G to package lentivirus particles. According to the enhanced green fluorescent protein （EGFP）expression, the functional titer of LV vector was determined by cell counting after transduction into 293T cells. Results A 2241bp specific DNA fragment was amplified by PCR. Gene sequencing demonstrated that sequence was identical to that as reported in GenBank. The eGFP can be stablely expressed in the 293T cells. Finally we have obtained the EZH2 lentivirus particles with the 2.1 ×10^8TU/ml functional titer. Conclusion We have successfully constructed mouse EZH2 gene Lentiviral vector. The functional titer of LV vector was 2.1 ×10^8TU/ml.