人MeCP2基因shRNA真核表达质粒的构建及鉴定  

Construction and identification of eukaryotic expression vectors expressing shRNA sections targeting human MeCP2

作　　者：郭玲玲[1] 段凤英[1] 邱小华[1] 

机构地区：[1]南昌大学第二附属医院呼吸内科,江西南昌330006

出　　处：《实用肿瘤杂志》2008年第3期229-233,共5页Journal of Practical Oncology

基　　金：江西省科学技术厅社会发展攻关项目(编号:20041095)

摘　　要：目的构建针对人甲基化结合蛋白MeCP2的shRNA真核表达质粒,稳定转染肺腺癌细胞A-549观察其对MeCP2表达的影响。方法根据MeCP2的mRNA序列,利用Ambion网上设计软件设计两条针对该基因不同靶点的shRNA干扰序列,克隆到线性化的质粒载体上,PCR、测序鉴定插入成功,且插入方向正确。转染A-549细胞,观察其在mRNA和蛋白水平对MeCP2的影响。结果成功构建两个MeCP2的shRNA真核表达质粒,并稳定转染到肺癌细胞A-549中,两个真核表达质粒在mRNA水平和蛋白水平对MeCP2有明显的抑制作用。结论人MeCP2的shRNA真核表达质粒成功构建及鉴定为进一步研究该蛋白的功能奠定了基础。Objective To construct two eukaryotic expression vectors expressing short hairpin （shRNA） sections targeting human MeCP2 and to observe their effects on MeCP2 gene expression in human lung cancer cell A-549. Methods Finding the sequence of human MeCP2 gene in Pubmed, the oligonucleotides of shRNA were desingned by the software of Ambion company, then sythesized and directionlly cloned into plasmid pSilencer-2. 1-U6 Hygro with Amp gene and hygro gene. The recombinant vectors were comfirmed by PCR and DNA sequencing analysis. The recombinant vectors were transfected into A-549 cell line,the effects on MeCP2 at mRNA and protein levels were observed. Results Two shRNA expressing vectors were recombined and transfected into A-549 cell line successfully. The mRNA and protein of MeCP2 were reduced visibly. Conclusion The construction of two eukaryotic expression vectors expressing shRNA sections targeting human MeCP2 and identification have successfully established a favourable foundation for further study on the function of MeCP2.

关 键 词：肺肿瘤/遗传学 基因表达 质粒 克隆 分子 肿瘤细胞 培养的 

分 类 号：R734.2[医药卫生—肿瘤]