靶向 Pokemon 基因 shRNA 重组质粒的构建及其在 CNE2细胞的筛选

摘要

Objective:To construct three short hairPin RNA( shRNA)recombinant Plasmids targeting human Pokemon gene and screen a Plasmid which can block the Pokemon exPression at maximal degree in nasoPharyngeal carcinoma( NPC)CNE2 cells. Methods:Three shRNAs were designed according to the coding sequences of the Pokemon gene. The shRNA sequences were inserted into PRNAT - U6. 1 / Neo during restriction enzyme,connection, transformation and DNA sequencing. The recombinant Plasmids and blank Plasmid were transfected into CNE2 cells using LiPofectamine 2000. The exPression of Pokemon mRNA and Protein was detected by real time Polymerase chain reaction(PCR)and Western blot,the transfected blank Plasmid CNE2 was used as a negative control,the untreated CNE2 was used as a blank control. Results:The results of DNA sequencing showed shRNA fragments were accordance with the designed sequences. ComParing with the negative control,the Pokemon mRNA and Protein exPression levels of the shRNA grouPs were down - regulated. The Pokemon results of shRNA2 grouP was lowest. Conclusion:Construc-tion and screening shRNA recombinant Plasmids targeting human Pokemon gene can Provide an effective tool for fur-ther studying the role of Pokemon gene in occurrence and develoPment of NPC.%目的：构建3个靶向 Pokemon 基因的短发夹 RNA（shRNA）真核表达载体，在鼻咽癌细胞株 CNE2中筛选出干扰效果最佳的载体。方法：依据 shRNA 设计原则，针对 Pokemon mRNA 序列设计并化学合成3对RNA 干扰序列，经酶切、连接和转化，插入 PRNAT - U6.1/ Neo 质粒，经 DNA 测序验证。脂质体介导 shRNA 重组质粒及空白质粒转染 CNE2细胞，荧光定量 PCR 和 Western blot 检测 Pokemon 在 mRNA 和蛋白水平的表达，转染空白质粒的 CNE2作为阴性对照，未加处理的 CNE2作为空白对照。结果：DNA 测序结果显示 shRNA片段与设计序列一致；转染各重组质粒后的 CNE2细胞，与阴性对照相比，Pokemon 基因在 mRNA 和蛋白水平均有下调表达，其中以 shRNA2重组质粒干扰 Pokemon 表达的作用最显著。结论：筛选出高效干扰 Pokemon表达的重组质粒 shRNA2，为研究 Pokemon 基因在鼻咽癌发生发展中的作用提供有效的研究工具。