人HMG盒转录因子1基因shRNA质粒的构建及其功能鉴定

Construction and function of shRNA plasmid for HMG-box transcription factor 1 gene

目的构建人HMG盒转录因子1(HMG-box transcription factor 1,HBP1)基因shRNA质粒,并探讨该基因的相关功能。方法合成HBP1基因干扰序列,将其插入至慢病毒表达载体pLL3. 7中,构建重组质粒pLL3. 7-shHBP1,经TurboFectTM脂质体介导转染HEK293T细胞,收集含病毒的培养基,分别感染HeLa及HepG2细胞,经puromycin持续筛选,获得稳定转染细胞株。Real-time PCR及Western blot法分别检测HeLa及HepG2细胞中HBP1基因m RNA转录及蛋白表达水平,MTT及流式细胞术分别检测HBP1基因抑制对HeLa细胞增殖及凋亡的影响。结果经测序鉴定,重组质粒pLL3. 7-shHBP1构建正确。HeLa及HepG2细胞中HBP1基因mRNA转录及蛋白表达水平均明显下调(P <0. 01);HBP1基因抑制后,HeLa细胞的增殖速度明显加快(P <0. 01),细胞凋亡明显减少(P <0. 01)。结论成功构建了HBP1基因shRNA质粒,抑制HBP1表达后可使HeLa细胞的增殖速度加快,细胞凋亡减少。本实验为HBP1基因相关功能的进一步研究奠定了基础。

Objective To construct a shRNA plasmid for HMG-box transcription factor 1(HBP1)gene and investigate its function. Methods The shRNA sequence of HBP1 gene was synthesized and inserted into lentivirus expression vector p LL3. 7. The constructed recombinant plasmid pLL3.7-shHBP1 was transfected to HEK293 T cells in mediation of liposome TurboFectTM. The medium containing virus was collected,with which HeLa and HepG2 cells were infected,and the cell strains transfected stably were obtained by continuously screening with puromycin. The mRNA transcription levels of mRNA in HeLa and HepG2 cells were determined by real-time PCR,while the protein expression levels by Western blot. The effect of HBP 1 gene suppression on the proliferation of HeLa cells was evaluated by MTT assay,while that on apoptosis by flow cytometry. Results Sequencing proved that recombinant plasmid pLL3. 7-shHBP1 was constructed correctly. After HBP1 gene suppression,both mRNA transcription and protein expression levels of HBP1 in HeLa and HepG2 cells were significantly down-regulated(P < 0. 01). The proliferation rate of HeLa cells increased significantly,while the apoptosis decreased significantly(each P < 0. 01). Conclusion The shRNA plasmid for HBP1 gene was successfully expressed,which laid a foundation of further study on the relevant function of HBP1 gene.