Red系统三种蛋白在哺乳动物细胞核中定位表达的设计和构建  

Design and Construction of Red Proteins Expressing in Mammalian Cell Nucleolus

作　　者：张晓楠[1] 黄勇[2] 曹蔚[1] 侯颖[1] 赵玮钦[3] 关璐媛[3] 陈苏民[3] 吴元明[4] 

机构地区：[1]第四军医大学药理教研室,陕西西安710032 [2]西北工业大学生命科学院,陕西西安710072 [3]第四军医大学生物化学与分子生物学教研室,陕西西安710032 [4]第四军医大学病理与生理学教研室,陕西西安710032

出　　处：《现代生物医学进展》2008年第10期1809-1812,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金资助项目(No.3017522)

摘　　要：目的:构建在哺乳动物细胞核中同时定位表达Red系统三种蛋白的载体。方法:通过给red基因(gam、bet和exo)的3,端分别加入由7个氨基(pro-lys-lys-lys-Arg-Lys-val)组成的核定位信号序列(nuclear localization signal,NLS)和4个甘氨酸构成的linker序列,再从λ噬菌体基因组中,通过PCR技术分别扩增到C端均都带有NLS的gam、bet和exo的DNA全长序列,将三种PCR产物依次克隆入真核表达载体pIRES2-EGFP中,得到含3个目的基因(gam-NLS、bet-NLs和exo-NLS)的三顺反子表达载体pCMV-gNIbNIxN,转染培养的哺乳类细胞后,用作者制备并纯化的抗Red3种蛋白的抗体进行间接免疫荧光检测,观察蛋白的定位情况。结果:不带有NLS的Red3种蛋白在细胞中表达主要集中在细胞质很少进入细胞核;而带有NLS的Red3种蛋白于不同时间转染人和鼠的两种细胞后,均表达定位于细胞核中。结论:三顺反子表达载体pCMV-gNIbNIxN的成功构建为进一步探索将λ噬菌体的red系统用于哺乳动物细胞的基因替代打下基础。Objective： To designed and constructed red system proteins of expressing and location in mammalian cell nucleolus. Methods Firstly, a seguence of oligonuceotide decoding nucleus localization signal （pro-lys-lys-lys-Arg-Lys-val） and linker were ligated to the 3,-terminus primer of three Red gene, Red -NLS recombinant encoding gene （gam -NLS, bet- NLS and exo -NLS ） were amplified from phage λ, genome by PCR. Secondly, three target genes were subcloned into expression vector plRES2-EGFP. So we obtained a tfi- cistron expression vector pCMV-gNIbNIxN, which could express three Red proteins at same time. Finally, it was transfered into eukaryotic cells MC3T3 and HeLa by transient transfection, and detected the localization of target proteins in cell by immunofluorescence technology. Results： Three Red protein were localized in cell nucleolus. Conclusion： This system will be helpful in further investigation of functions of red gene.

关 键 词：Red蛋白 核定位信号 同源重组系统 蛋白质表达 

分 类 号：Q786[生物学—分子生物学]