JTV1基因siRNA重组表达质粒的构建及其对K562细胞增殖的影响  

Construction of siRNA expression vector for JTV1 gene and its effect on proliferation of K562 cells

作　　者：吴燕[1] 刘北忠[1,2] 王翀 钟梁[2] 朱丹[2] 王春光[2] 黎亮[2] 高艳军[2] 

机构地区：[1]重庆医科大学附属永川医院中心实验室,重庆400016 [2]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2012年第2期148-151,156,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30300449);国家中医药管理局(02-03ZP52);重庆医科大学课题(XBYB2007104)

摘　　要：目的构建JTV1基因siRNA重组表达质粒,转染K562细胞,鉴定其干扰作用。方法人工合成靶向JTV1基因的siRNA干扰序列,克隆至载体pGeneSil-1上,构建重组表达质粒pGeneSil-1-JTV1-1.1 siRNA,转染人K562细胞,经G418稳定筛选后,采用RT-PCR及Western blot法分别检测重组表达质粒对K562细胞中JTV1基因转录和翻译的影响;MTT法检测抑制JTV1基因的表达对K562细胞增殖的影响。结果重组表达质粒pGeneSil-1-JTV1-1.1 siRNA经测序证明构建正确;其转染K562细胞后,细胞JTV1基因的转录和翻译水平均明显降低;抑制JTV1的表达可促进K562细胞增殖。结论已成功构建了pGeneSil-1-JTV1-1.1 siRNA质粒,并获得了稳定的JTV1基因表达受抑的K562细胞克隆,为进一步研究JTV1基因的功能及其与肿瘤的相关性奠定了基础。Objective To construct the siRNA expression vector for JTV1 gene, transiect to K562 cells and identity its interference effect. Methods The siRNA interfering sequence targeting to JTV1 gene was synthesized and cloned into vector pGeneSil-1. The constructed recombinant plasmid pGeneSil-l-JTVl-1. 1 siRNA was transfected to human K562 cells. The positive clones were screened with G418, based on which the effect of recombinant plasmid pGeneSil-l-JTVl-1. 1 siRNA on transcription and translation of JTV1 gene in K562 cells was investigated by RT-PCR and Western blot, and that of JTV1 gene expression on proliferation of K562 cells by MTY method. Results Sequencing proved that recombinant plasmid pGeneSil-l-JTVl-1. 1 siRNA was constructed correctly. Both the transcription and translation levels of JTV1 gene in K562 cells transfected with the recombinant plasmid decreased significantly. The inhibition of JTV1 expression promoted the proliferation of K562 cells. Conclusion Recombinant plasmid pGeneSil-l-JTVl-1. 1 siRNA was successfully constructed, and stable K562 cell clones in which the expression of JTV1 gene was inhibited were obtained, which laid a foundation of further study on function of JTV1 gene and its correlation to tumor.

关 键 词：JTV1基因 RNA干扰 K562细胞 

分 类 号：Q782[生物学—分子生物学] R73-362[医药卫生—肿瘤]