pApoptin-EGFP真核表达载体的构建及其对膀胱肿瘤T24的促凋亡作用

Construction of eukaryotic expression plasmid pApoptin-EGFP and the apoptosis induction of it on bladder tumor cell line T24

目的构建真核表达质粒pApoptin-EGFP并探讨其对人膀胱肿瘤细胞株T24促凋亡作用。方法采用PCR的方法从质粒p3×flag-Apoptin-myc扩增出野生Apoptin片段,将其定向克隆于pEGFP-N1载体的EcoRⅠ和BamHⅠ酶切位点,构建能表达Apoptin-EGFP融合蛋白的真核表达质粒pApoptin-EGFP,酶切、测序鉴定,RT-PCR、荧光显微镜分析Apoptin基因表达,脂质体介导瞬时转染膀胱肿瘤T24细胞,流式细胞仪检测调亡及细胞周期变化。结果成功构建pApoptin-EGFP重组质粒,并在被转染细胞中检测到Apoptin基因表达;瞬时转染后不同时相点均可检测到细胞凋亡,与空质粒组相比具有显著差异,细胞周期表现为G1/G0期阻滞。结论成功构建pApoptin-EGFP真核表达质粒,重组质粒能在T24细胞中顺利表达Apoptin-EGFP融合蛋白,重组质粒转染可诱导T24细胞凋亡并阻滞细胞周期于G1/G0期。

Objective To construct the eukaryotic expression plasmid pApoptin-EGFP and investigate its apoptosis effects on bladder tumor cell line 124. Methods Gene of wild-type Apoptin was amplified from the plasmid p3 × flag-Apoptin-myc,and the product was directed cloned into vector pEGFP-N1 between the enzyme sites of EcoRⅠ and BamH Ⅰ . The recombinant plasmid was identified by enzyme digesting and nucleotide sequencing. Then the recombinant plasmid was transfected into T24 cells with Lipofectamine 2000; the expression of apoptin gene was detected by RT-PCR and fluorescence microscope. Transfection efficiency, apoptosis, and the cell cycle of T24 were analyzed by using flow cytometry. Results The recombinant plasmid pApoptin-EGFP had been constructed correctly and the expression of apoptin gene could be detected in T24 cells. Apoptosis of T24 cells could be detected post transient transfection, while the apoptosis rate of T24 cells which transfected with pApoptin-EGFP was much higher than that of control and cell cycle G1/G0 arrest was found either. Conclusion The eukaryotic expression plasmid pApoptin-EGFP is successfully constructed and it could correctly express the fusion protein in T24 cells. Transfection with plasmid pApoptin-EGFP could induce cell apoptosis and cell cycle G1/G0 arrest on T24 cells.