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目的构建4个新靶点的人细胞周期蛋白依赖性激酶2(CDK2)干扰RNA真核表达载体,转染人脑胶质瘤细胞后,检测出干扰效果最好的载体及细胞增殖能力的变化。方法构建4个新靶点CDK2干扰RNA真核表达载体并用双酶切和测序鉴定;分别转染上述4个载体到人脑胶质瘤细胞株SHG44;通过逆转录聚合酶链反应(RT-PCR)比较转染后CDK2 mRNA的表达量,选出干扰效果最好的一个,检测细胞增殖能力的变化。结果成功构建4个新靶点的CDK2干扰RNA真核表达载体PCDK2-1、PCDK2-2、PCDK2-3、PCDK2-4;CDK2 mRNA表达和细胞增殖明显受到抑制,PCDK2-1的干扰效果为56%;PCDK2-1-SHG44细胞与对照组相比增殖能力减弱。结论成功构建并筛选出效果最好的新靶点CDK2干扰RNA真核表达载体,并使SHG44细胞的增殖水平降低。
Objective To construct a new target eukaryotic expression vector of human cyclin dependent kinase 2 (CDK2) interfering RNA targeting 4 new targets. After transfection with human glioma cells, the best interfering vector and cell proliferative capacity were detected . Methods Four novel target CDK2 RNAi eukaryotic expression vectors were constructed and identified by double enzyme digestion and sequencing. The four vectors were transfected into human glioma cell line SHG44, respectively. The expression of CDK2 was detected by reverse transcriptase-polymerase chain reaction ) Compare the expression of CDK2 mRNA after transfection and select the one with the best interference effect to detect the change of cell proliferation ability. RESULTS: Four new target sites for CDK2 interference RNA were successfully constructed. The expression of PCDK2-1, PCDK2-2, PCDK2-3 and PCDK2-4, CDK2 mRNA and cell proliferation were significantly inhibited. The interference effect of PCDK2-1 was 56 %; PCDK2-1-SHG44 cells compared with the control group decreased proliferation. Conclusion The CDK2 interfering RNA eukaryotic expression vector was successfully constructed and screened, and the proliferation of SHG44 cells was reduced.