RNAi表达载体干扰四型肽精氨酸脱亚氨基酶基因在HL-60细胞系中的表达  

The expressive vector of RNA interference can effect the expressions of PADI-4 gene in HL-60 cells

作　　者：张慧[1] 范列英[1] 卢天宝[1] 宗明[1] 吕红根[1] 杨蔺[1] 

机构地区：[1]上海同济大学附属东方医院检验科,上海200120

出　　处：《中国免疫学杂志》2008年第11期972-975,共4页Chinese Journal of Immunology

基　　金：上海市卫生局科技发展基金项目(044100)

摘　　要：目的:设计针对PADI-4基因的RNAi表达载体,抑制PADI-4基因在HL-60细胞中的表达,初步确定PADI-4基因在类风湿性关节炎发生发展中的作用,为后续研究奠定实验基础。方法:设计并合成4对针对PADI-4基因编码区的核苷酸链,核苷酸链经体外退火后形成双链SiRNA,克隆进入pSiRNA-hH1neo G2空载体后转化入GT116细胞,通过蓝白斑筛选后,挑取白斑进行扩增,抽提质粒并对重组质粒进行双酶切分析。以脂质体转染法,分别将pSiRNA-hH1neo G2空载体和4个重组质粒载体转染进入HL-60细胞系,于转染的第3、5、7、10、14天后收集细胞,抽提细胞总RNA后,反转录成cDNA,用Real-time PCR技术检测各实验组HL-60细胞内PADI-4基因mRNA水平的表达情况,并做统计分析。结果:经Acc65Ⅰ和HindⅢ双酶切鉴定,重组质粒中已经插入目的片段,为合格质粒;转染pSiRNA表达载体后,HL-60细胞系中PADI-4基因mRNA表达水平下降,其中以第3号载体的干扰效果最优,干扰效率达到62%。结论:成功构建针对PADI-4基因的pSiRNA表达载体,并且筛选出干扰效率最好的载体,为后续研究PADI-4基因在类风湿性关节炎中的作用奠定实验基础。Objective： To prove the effect of PADI-4 gene in the development of rheumatoid arthritis. Methods： Four SiRNA sequences were designed for PADI-4 gene,and the SiRNAs were cloned into blank pSiRNA-hh1neo G2 vectors. The vectors were transformed into GT116 E. coli competent ceils. By white-blue selection system, the fight vectors were gotten. After transfection into HL-60 cells, the cells were collected on 3,5,7,10 and 14 day,the levels of PADI-4 mRNA were detected by Real-time PCR.Results：Digestion by Acc 65 I and Hind Ⅲ ,the recombinant expressive vector of RNA interference was obtained successfully. The PADI-4 mRNA generated by the ceils transfected with the vector of SiRNAs were reduced, and the level was not change in normal cells. Conclusion：The recombinant expressive vector of RNA interference is obtained successfully and the recombinant expressive vector can affect expression of PADI-4 gene in HL-60 cells.

关 键 词：PADI-4基因 SIRNA RNA干扰 HL-60细胞系 

分 类 号：R3[医药卫生—基础医学]