核磷蛋白NPM1的RNA干扰慢病毒载体的构建及鉴定简

目的：构建高效抑制核磷蛋白NPMl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p113．7；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-9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HTl080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HTl080细胞，RT-PCR和Western印迹检测分别在RNA和蛋白质水平证实NPMl的表达显著降低。结论：构建的RNA干扰载体pll-shRNA2能有效抑制NPMl的表达，为NPMl功能的研究提供了有力工具。

Construction and Identification of Interference Vector Targeting Nu cleophosmin 1 Gene

Objective： To construct a high effective short hairpin RNA（shRNA） interference vector of nucleophos min I（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 p113.7. 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 ceils, 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 p113.7 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 NPM 1 function.