红细胞分化相关因子EDRF1的反义RNA下调蛋白激酶C-α mRNA的表达  

Antisense RNA of a novel erythroid related gene EDRF1 down-regulates

作　　者：王敦成[1] 张晓东[2] 王建安[1] 赖春宁[1] 黎燕[1] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]中国医学科学院基础医学研究所,北京100005

出　　处：《生物技术通讯》2001年第3期171-174,共4页Letters in Biotechnology

摘　　要：为了研究通过功能筛选得到的一个新的红细胞分化相关的全长cDNA(命名为EDRF1)的功能 ,选择K5 6 2细胞作为模型细胞来观察反义EDRF1表达对细胞功能的影响。通过快速构建 ,同时得到了正向和反向插入片段的真核表达载体pcDNA3 EDRF1 S(sense)及pcDNA3 AS(antisense) ,并通过改进的LipofectAMINE细胞转染和G418筛选 ,得到了稳定表达的细胞株 ,进行基因组PCR鉴定 ,证明了转染的高效性。NorthemBlot试验的结果表明 ,反义载体转染的K5 6 2细胞中 ,EDRF1在mRNA表达水平上受到下调 ,提示EDRF1反义DNA的表达可能抑制了EDRF1mRNA的正常转录。进一步 ,γ 珠蛋白和PKC α的表达在反义构建质粒转染K5 6 2细胞中的表达均受到下调 ,这可能传递了红细胞分化的负反馈信号 。In the previous study, the monoclonal antibodies were utilized, which was corresponding to HS2-binding proteins, to screen λ-gtll human cDNA expression library of fetal liver. In this way, two cDNA clones named EDRF1 and EDRF2 were obtained respectively. This study focuses on the function of erythroid related factor 1, which was identified that it owns a whole sequence, by transfecting K562 cells characterized by stable expression from the evidence of genomic integration of Neo gene by G418 selection. EDRF1 mRNA expression was suppressed in pcDNA3 antisense construct plasmid trasfected K562 cells identified by Northern blot. It suggests that antisense RNA expression disturbed that synthesis of EDRF1 mrna efficiently. Both γ-globin and PKC-α mRNA expressions were down-regulated in antisense construct plasmid transfected K562 cells demonstrated by semiquantitative RT-PCR. It suggests that the inhibition of PKC signal may be the negative modulation of K562 differentiation and globin synthesis.

关 键 词：反义RNA 红细胞分化 蛋白激酶C-Α 信号转导 γ- 珠蛋白 表达 

分 类 号：Q78[生物学—分子生物学] Q254