摘要
目的构建基于人源性miRNA-30,针对EGFP基因的RNAi表达框架并验证其有效性。方法融合PCR构建RNAi表达框架,经凝胶电泳、测序比对及二级结构预测后,将该表达框架与p EGFP-N2质粒共转染A549、HCT116及HEK-293细胞,48 h后倒置荧光显微镜观察EGFP的表达情况并用流式细胞仪检测平均荧光强度。结果凝胶电泳和测序比对均显示RNAi表达框架与设计的相符,二级结构预测显示,新型RNAi表达框架与人源性miRNA-30的二级结构高度相似;共转染该表达框架与p EGFP-N2质粒48 h后,倒置荧光显微镜观察结果显示,3种细胞的荧光强度均明显减弱,流式细胞仪检测结果显示,3种细胞中共转染组与单独转染组相比平均荧光强度的差异均有统计学意义(P<0.05)。结论新型RNAi表达框架构建成功,且能有效干扰EGFP在A549,HCT116和HEK-293细胞中的表达。
Objective To construct the expression cassettes of RNAi-based on the structure of human miRNA-30 and targeting EGFP gene, and validate its inhibition effect. Methods The expression cassettes of RNAi were synthesized by overlap extension PCR, and cotransfected into A549, HCT116 and HEK-293 cells with plasmid pEGFP-N2. Following agarose gel electrophoresis, DNA sequencing and secondary structure prediction, the expression of EGFP was observed by inverted fluorescence microscope and the mean fluorescence intensity was detected by flow cytometry after 48 hours. Results Agarose gel electrophoresis and DNA sequencing demonstrated that the expression cassettes of RNAi were constructed correctly, and the results of structure prediction showed that the secondary structure of the novel expression cassettes of RNAi was highly similar with that of human miRNA-30. After co-transfection with the expression cassettes and plasmid pEGFP-N2, the fluorescence intensities of the 3 different cell lines were significantly reduced under inverted fluorescence microscope, and the results of flow cytometry showed that the mean fluorescent intensities between co-transfection group and individual transfection group in the 3 cells were significantly different （P 〈 0.05 ）. Conclusion The novel expression cassettes of RNAi were constructed successfully, which may effectively disturb the expression of EGFP protein in A549, HCTI16 and HEK-293 ceils.
出处
《临床检验杂志》
CAS
CSCD
2016年第8期625-629,共5页
Chinese Journal of Clinical Laboratory Science
基金
中南大学研究生科研创新项目(2016zzts500)
中南大学本科生自由探索项目(201610533579)
