Survivin反义RNA真核表达载体的构建

目的构建Survivin反义RNA表达载体,从基因水平靶向封闭Survivin功能,为探讨喉癌细胞凋亡和增殖的影响奠定基础。方法将Survivin大部分编码序列cDNA片段反向插入pEGFPC1载体,构建Survivin反义RNA表达载体。用脂质体转染技术将重组质粒转染人喉癌细胞株Hep2,并利用G418(300mg/ml的维持浓度)筛选,获得稳定细胞株(命名为HpEGFP/Survivin)。用蛋白免疫印迹Westernblot从蛋白质水平检测反义SurvivinRNA封闭Survivin的表达效果。结果构建的Survivin反义RNA表达载体(命名为pEGFP/Survivin),经酶切电泳和测序证实碱基序列的正确性。蛋白免疫印迹Westernblot结果表明,构建的Survivin反义RNA表达载体转染人喉癌细胞株Hep2,从蛋白水平抑制了Survivin的表达,抑制率为30%。说明反义SurvivinRNA载体构建成功。结论构建的Survivin反义RNA表达载体从蛋白水平抑制了Survivin的表达,可以进一步用于抑制Survivin基因表达的实验研究。

Construction of antisense survivin eukaryotic expression plasmid

Objective To construct antisense survivin expression vector related to the study of antisense survivin gene expression. Methods Survivin cDNA was inserted into the expression plasmid pEGFP-C1 site in an antisense orientation to produce antisense survivin expression vector. The electrophoresis and sequencing were carried out. Then it was transfected to human laryngeal carcinoma cell line Hep-2 by lipofectamine. HpEGFP/Survivin cells were obstained by using G418. The levels of protein before and after transfection were determined by Western-blot. Results The antisense vector were verified by the electrophoresis and sequencing. The expression of survivin was inhibited after recombination antisense survivin plasmid were transfected. The level of protein was inhibited. The rate of inhibitor was 30%. Conclusion The recombination antisense survivin plasmid can partly inhibit the expression of survivin and can be used for related study of antisense gene expression.