IKCa1基因shRNA真核表达载体的构建及其在HeLa细胞中的表达

Construction of shRNA eukaryotic expression vectors of IKCa1 gene and its expression in HeLa cells

目的:构建针对钙激活性中电导钾离子通道(intermediate-conductance Ca2+-activated K+channels,IKCa1)的小发夹RNA(small hair RNA,shRNA)真核表达载体,观察其在宫颈癌细胞HeLa中的表达。方法:设计、合成IKCa1基因特异性shRNA序列,插入空载体pGenesil-1.1中,构建重组体,脂质体法转染宫颈癌HeLa细胞,半定量RT-PCR和Western blotting技术检测转染前后HeLa细胞中IKCa1的表达。结果:成功构建IKCa1 shRNA真核表达载体,转染后HeLa细胞中IKCa1的表达受到显著抑制。结论:成功构建IKCa1特异性shRNA表达载体,为进一步研究IKCa1参与宫颈癌发生发展的机制奠定实验基础。

Objective： To construct eukaryotic expression vectors of RNA interference of IKCa1 gene and observe its expression in HeLa cells of cervical cancer.Methods： shRNAs which specifically targeted for the sequences of IKCa1 gene were designed and synthesized.The shRNAs were cloned into the pGenesil-1.1 vector.HeLa cells were transfected with the recombinant plasmids,RT-PCR and Western blotting were used to certify the expression of IKCa1.Results： The eukaryotic expression vectors of RNA interference of IKCa1 gene were constructed successfully.The expression of IKCa1 gene in HeLa cells were efficiently and specifically inhibited after transfected with the recombinant plasmids.Conclusion：The eukaryotic expression vectors of RNA interference of IKCa1 gene are constructed successfully.