血液肿瘤中NPM1突变基因的载体构建及表达分析  

Vector Construction and Expression Analysis of NPM1 Mutant Gene in Hematological Tumors

作　　者：彭晨阳 任安琦 李婉蓉 武福云 陈义芝 PENG Chenyang;REN An-qi;LI Wan-rong;WU Fu-yun;CHEN Yi-zhi(Department of Pathophysiology,College of Basic Medicine,Hubei University of Medicine,Shiyan,Hubei 442000,China)

机构地区：[1]湖北医药学院基础医学院病理生理学教研室,湖北十堰442000

出　　处：《湖北医药学院学报》2023年第1期11-14,F0002,共5页Journal of Hubei University of Medicine

基　　金：湖北医药学院研究生启动金项目(2020QDJZR026);大学生创新创业训练计划项目(S202110929009)。

摘　　要：目的:NPM1基因突变导致多种类型的血液肿瘤,其中最常见的是NPM1突变导致急性髓系白血病(AML)及间变性大细胞淋巴瘤(ALCL)。构建这两种最常见的NPM1突变基因真核表达载体并分析其表达情况,对于研究NPM1突变与血液肿瘤发生的关系具有重要意义。方法:设计NPM1突变基因引物,通过重叠PCR扩增基因片段,并插入到pEGFP-N1载体,构建GFP-NPMc+及GFP-NPM-ALK真核表达载体;转染GFP-NPM1突变载体到HEK293T细胞,荧光显微镜观察突变蛋白细胞定位情况;免疫印迹检测NPM1突变蛋白过表达对相关细胞信号通路的影响。结果:双酶切实验显示GFP-NPMc+和GFP-NPM-ALK真核表达载体构建成功;荧光显微镜观察显示NPMc+和NPM-ALK突变蛋白均主要定位于细胞质;免疫印迹结果显示NPM1突变能够激活PKC和ERK等信号通路。结论:成功构建了血液肿瘤中最常见的NPM1突变基因表达载体,并通过NPM1蛋白细胞定位的分析,我们认为NPM1突变蛋白共有的组成结构域NPM-N端可能在各种血液肿瘤发生中发挥重要作用。Objective NPM1 gene mutations lead to many types of hematological tumors,the most common of which is NPM1 mutations leading to acute myeloid leukemia(AML)and anaplastic large cell lymphoma(ALCL).The construction of the eukaryotic expression vectors of the two most common NPM1 mutations and the analysis of their expression are of great significance for the study of the relationship between NPM1 mutations and the occurrence of blood tumors.Methods NPM1 mutant gene primers were designed,gene fragments were amplified by overlapping PCR,and inserted into pEGFP-N1 vector to construct GFP-NPMc+and GFP-NPM-ALK eukaryotic expression vectors.The GFP-NPM1 mutant vector was transfected into HEK293T cells,and the cellular localization of the mutant protein was observed by a fluorescence microscopy.Immunoblotting was used to detect the effect of overexpression of NPM1 mutant protein on related cell signal pathways.Results The double enzyme digestion experiment showed that GFP-NPMc+and GFP-NPM-ALK eukaryotic expression vectors were constructed successfully.Fluorescence microscopy showed that both NPMc+and NPM-ALK were localized in the cytoplasm;Immunoblotting results showed that NPM1 mutation could activate PKC and ERK signaling pathways.Conclusion The most common NPM1 mutant gene expression vector in hematological tumors has been successfully constructed,and through the analysis of the cellular localization of NPM1 protein,we believe that the NPM-N-terminal domain,which is common to NPM1 mutant proteins,may play an important role in the occurrence of various hematological tumors.

关 键 词：NPM1 血液肿瘤 载体构建 蛋白表达 

分 类 号：R733[医药卫生—肿瘤] Q78[医药卫生—临床医学]