摘要
目的构建含有EGFP及Ad5.EIA基因的重组真核表达载体pEGFP-E1A,研究其在体外对肝癌细胞的裂解作用。方法对pUC119-E1A质粒双酶切获得E1A基因,定向克隆带EGFP和neo筛选标记的真核表达载体到pEGFP-C1载体中,构建pEGFP-E1A质粒,通过脂质体导入人肝癌细胞SMMC-7721中,经RT-PCR及间接免疫荧光方法确认EIA基因的导入,DNAladder法检测EIA基因对SMMC-7721细胞的凋亡诱导作用。结果酶切和序列测定结果显示重组质粒构建正确,RT-PCR及间接免疫荧光结果显示,外源性E1A基因已整合于SMMC-7721细胞并获稳定表达,在6小时就导致SMMC-7721出现细胞凋亡。结论初步证实E1A基因在体外对人肝癌SMMC-7721细胞具有诱导细胞凋亡的作用,为进一步探索Ad5.EIA基因的抗瘤作用提供实验基础。
Objective To construction of recombinant eukaryotic expression vector pEGFP-E1A containing EGFP and Ad5.EIA,in vitro study of inhibition effect to SMMC-7721.Methods The E1A gene was digested from pUC119-E1A plasmid and cloned into pEGFP-C1,a eukaryotic expression vector containing EGFP and neo as selection marker,to form pEGFP-E1A.The pEGFP-E1A was transfected into SMMC-7721 hepatoma cell lines with lipofectin by identification with RT-PCR and immunofluorescence assay.Apoptosis was evaluated using DNA gel electrophoresis.Results Enzyme restriction and sequencing data indicated that the recombinant vector was constructed exactly,RT-PCR and immunofluorescence assay result shown that the pUC119-E1A recombinant eukaryotic expression vector can efficiently express E1A in SMMC-7721 cells,and apoptosis of SMMC-7721 appears at 6 hours.Conclusion The results preliminarily confirmed E1A gene can induced apoptosis SMMC-7721 human hepatoma cell in vitro,which offered the basic experimental conditions for exploring the antitumor activity led by the Ad5.EIA gene.
出处
《中国实验诊断学》
北大核心
2010年第12期1906-1910,共5页
Chinese Journal of Laboratory Diagnosis
