Prelamin A相互作用蛋白Narf的鉴定和表达分析  

Identification and Expression Analysis of Narf, an Interacting Protein of Prelamin A

作　　者：陈维春[1,2] 刘振杰[2,3] 蒋智文[1] 彭琪[1] 袁源[1] 郑慧玲[1,2] 刘新光[1,2] 

机构地区：[1]广东医学院衰老研究所,广东东莞523808 [2]广东医学院生物化学与分子生物学研究所,广东湛江524023 [3]广东省中医院检验科,广东广州510120

出　　处：《生物技术》2011年第5期22-27,共6页Biotechnology

基　　金：国家自然科学基金项目(No.30871440;30900739);广东省自然科学基金项目(7301506和9252402301000002);广东省高校自然科学研究重点项目(06Z015);广东省医学科研基金项目(B2007138);东莞市科技计划项目(2008108101062)资助

摘　　要：目的:探讨A型核纤层蛋白前体(prelamin A)在细胞内堆积造成细胞早老的机理,筛选了prelamin A相互作用蛋白并研究其在早老细胞中的表达情况。方法:以prelamin A的C末端区域为诱饵蛋白,采用酵母双杂交方法从人骨骼肌cDNA文库中筛选prelamin A相互作用蛋白。构建了prelamin A识别因子(Narf)与绿色荧光蛋白融合表达载体pEGFP-Narf,与红色荧光蛋白-prelamin A融合表达质粒pDsRed-PLA共转染HEK293细胞,激光共聚焦显微观察共定位情况。Western blotting检测Narf在衰老表型HEK293PLA细胞的表达情况。结果:筛选得到包括Narf在内的7个候选相互作用蛋白。Narf与prelamin A能相互作用并共定位于核纤层,在prelamin A过表达的HEK293PLA细胞中Narf表达没有升高。结论:Narf在细胞内与prelamin A相互作用,且表达量不受后者影响。Objective：In order to reveal the mechanism of cell aging resulted from accumulation of prelamin A,proteins interacted with prelamin A were screened from human skeletal muscle library and the expression of nuclear prelamin A recognition factor（Narf） was analysized.Method：A bait plasmid pGBKT7-PLA-C was constructed containing C-teminal amino acids 389-664 of prelamin A and cotransformed into AH109 yeast competent cells with human skeletal muscle library.Narf gene was inserted into eukaryotic vector pEGFP generating the recombinant vector pEGFP-Narf,which was co-transfected into HEK293 cells with pDsRed-PLA and visualized by laser confocal microscopy.Expression of Narf in HEK293PLA cells was identifed by Western blotting.Result：Seven candidate proteins that interacted with prelamin A were identified by yeast two-hybrid screening including Narf.Colocalization assay showed that Narf and prelamin A were co-located around nucleus of HEK293 cells.The expression of Narf was not increased in the HEK293PLA cells,in which prelamin A was over expressed.Conclusion：Narf was able to interact with prelamin A and the latter can＇t affect Narf＇s expression.

关 键 词：酵母双杂交 A型核纤层蛋白前体 衰老 共定位 

分 类 号：Q786[生物学—分子生物学]