人EFTUD2基因靶向小分子化合物筛选细胞模型的建立*

目的 体外建立一个以荧光素酶为报告基因,靶向人EFTUD2基因的化合物筛选细胞模型。方法 应用限制性内切酶双酶切LV6载体,将人EFTUD2pro-0.5kb启动子和荧光素酶报告基因的融合片段重组入LV6慢病毒载体。以重组慢病毒感染HepG2细胞,采用嘌呤霉素筛选和有限稀释法获得Epro-LUC-HepG2细胞株。利用荧光素酶报告基因系统挑选最佳单克隆细胞株并扩大培养。结果 准确构建了LV6-Epro0.5-LUC慢病毒载体,并成功筛选出可稳定表达荧光素酶的Epro-LUC-HepG2单克隆细胞株。根据荧光素酶报告基因检测结果,挑选了最佳克隆株细胞,被命名为Epro-LUC-HepG2细胞。结论 我们成功构建了以人EFTUD2基因为靶点的Epro-LUC-HepG2细胞化合物筛选模型,有助于后续筛选新型靶向免疫调节药物。

Establishment of a cell model for screening human EFTUD2 gene-targeting small molecule compounds

Objective This paper aimed to establish a cell model with luciferase as reporter gene for screening human EFTUD2 gene-targeting compounds. Methods After double-digestion of LV6 lentivirus vector by restrictive enzymes, the target fragment was recombined into the LV6 lentivirus vector.The HepG2 cells were further infected with the recombinant lentivirus, and the Epro-LUC-HepG2 cell line was obtained by puromycin screening and limited dilution. By using the luciferase reporter gene system, we select the best single clone cell lines and expand the culture. Results The LV6-Epro0.5-LUC lentivirus vector was successfully constructed, and the Epro-LUC-HepG2 monoclonal cell lines expressing luciferase was successfully screened by lentivirus transfection and puromycin selection. According to the results of luciferase reporter gene detection, the best monoclonal cells were selected and named as Epro-LUC-HepG2 cells. Conclusion The Epro-LUC-HepG2 cells, a compounds screening model of targeting human EFTUD2 gene, is successfully constructed, which laid a hope for further screening of new targeted immunoregulatory agents.