CAJ | 학술논문

目的：构建高效抑制核磷蛋白NPM1基因的短发夹RNA（shRNA）干扰载体。方法：以人NPM1基因为靶序列，设计并合成shRNA序列，将其连入RNA干扰慢病毒载体pll3.7；酶切鉴定插入shRNA序列片段的质粒，经测序正确后转染293T细胞；Western印迹检测得到抑制效果好的载体pll-shRNA，将其与慢病毒载体共转染293T细胞，进行病毒的包装，将得到的病毒感染HT1080细胞，通过RT-PCR、Western印迹等方法验证其抑制效果。结果：酶切证实构建的载体pll-shRNA中已插入外源基因片段，转染293T细胞后都有抑制效果，其中pll-shRNA2的抑制效果最好；用pll-shRNA2病毒感染HT1080细胞，RT-PCR和Western印迹检测分别在RNA和蛋白质水平证实NPM1的表达显著降低。结论：构建的RNA干扰载体pll-shRNA2能有效抑制NPM1的表达，为NPM1功能的研究提供了有力工具。
목적：구건고효억제핵린단백NPM1기인적단발협RNA（shRNA）간우재체。방법：이인NPM1기인위파서렬，설계병합성shRNA서렬，장기련입RNA간우만병독재체pll3.7；매절감정삽입shRNA서렬편단적질립，경측서정학후전염293T세포；Western인적검측득도억제효과호적재체pll-shRNA，장기여만병독재체공전염293T세포，진행병독적포장，장득도적병독감염HT1080세포，통과RT-PCR、Western인적등방법험증기억제효과。결과：매절증실구건적재체pll-shRNA중이삽입외원기인편단，전염293T세포후도유억제효과，기중pll-shRNA2적억제효과최호；용pll-shRNA2병독감염HT1080세포，RT-PCR화Western인적검측분별재RNA화단백질수평증실NPM1적표체현저강저。결론：구건적RNA간우재체pll-shRNA2능유효억제NPM1적표체，위NPM1공능적연구제공료유력공구。
Objective: To construct a high effective short hairpin RNA(shRNA) interference vector of nucleophos-min 1(NPM1) gene. Methods: The shRNA oligonucleotide sequences were designed and synthesized according to human NPM1 gene sequence and the synthesized sequences were annealed to form double-strand oligonucleotide and cloned into interference vector plasmid pll3.7. The plasmids were restriction enzyme digested and sequenced for confirmation. The shRNA interference vectors were transfected into 293T cells. The shRNA interference effects of these vectors were detected by Western blotting. The shRNA vector with the best interference effect was and packaged into lentivirus. After infection into HT1080 cells, the interference effect was verified by RT-PCR and Western blotting. Results: NPM1 gene specific oligonucleotide sequences were successfully cloned into shRNA in-terference vectors pll3.7 and the expected vectors confirmed by restriction enzyme digestion and sequencing. It was found that the pll-shRNA2 vector had the highest interference effect on NPM1 compared with other vectors. After packaging into lentivirus, pll-shRNA2 was infected into HT1080 cells. It was shown that pll-shRNA2 also could significantly suppress NPM1 expression shown by the RT-PCR and Western blot results. Conclusion: The con-structed pll-shRNA2 can effectively inhibit the expression of NPM1, which provided a powerful method for investi-gating NPM1 function.