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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。
Objective To construct Survivin antisense RNA expression vector and to target the function of blocking Survivin gene from the level of gene, so as to lay the foundation for the study on the effect of Survivin on apoptosis and proliferation. Methods The cDNA fragment of most of the coding sequence of Survivin was inserted into pEGFPC1 vector reversely to construct antisense RNA expression vector of Survivin. The recombinant plasmids were transfected into human laryngeal carcinoma cell line Hep2 by liposome transfection and screened by G418 (the maintenance concentration of 300mg / ml) to obtain a stable cell line (named as HpEGFP / Survivin). The expression of antisense Survivin RNA blocking Survivin was detected from the protein level by western blot using Western blotting. Results The constructed Survivin antisense RNA expression vector (named pEGFP / Survivin) was verified by restriction enzyme digestion and sequencing. Western blot analysis of Western blotting results showed that Survivin antisense RNA was transfected into human laryngeal carcinoma cell line Hep2 and inhibited Survivin expression at a protein level of 30%. The antisense Survivin RNA vector was constructed successfully. Conclusion The constructed Survivin antisense RNA expression vector inhibits the expression of Survivin from the protein level and can be further used to inhibit Survivin gene expression.