靶向Pokemon基因shRNA重组质粒的构建及其在CNE2细胞的筛选

Construction of short hairpin RNA recombinant plasmids targeting human Pokemon gene and screening in CNE2 cells

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。

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.