人卵泡抑素基因短发夹RNA表达质粒的构建及其干扰效果的初步鉴定  

Construction and preliminary identification of interference efficiency of FS gene shRNA expression plasmid

作　　者：莫毅[1] 梁方方[2] 陈月凤 江如兰[1] 叶健[1] 谢丹尼[1] 

机构地区：[1]广西壮族自治区人口和计划生育研究中心,南宁530021 [2]广西壮族自治区畜牧研究所 [3]广西壮族自治区医科大学研究生院

出　　处：《中国计划生育学杂志》2013年第4期231-233,共3页Chinese Journal of Family Planning

摘　　要：目的:构建人卵泡抑素(FS)基因短发夹RNA(shRNA)真核表达质粒,并瞬时转染方式初步鉴定其干扰效果。方法:依据GenBank数据库提供的人FS基因mRNA核苷酸序列,利用Ambion网站上siRNA软件设计shRNA干扰靶序列,将该序列克隆到线性化的pLKO.1质粒载体上,构建pLKO.1-FS-shRNA重组质粒,并进行酶切和测序鉴定。确认质粒构建成功后,用脂质体(Lipofectamine TM 2000)将重组质粒瞬时转染3AO人卵巢癌细胞系,并用实时定量反转录PCR法检测重组质粒对FS基因的表达抑制效果。结果:构建的shRNA序列经DNA测序证实与设计序列完全一致;实时定量反转录PCR结果显示pLKO.1-FS-shRNA重组质粒瞬时转染的3AO人卵巢癌细胞后,细胞中的FS基因转录受到抑制,抑制率达59%。结论:成功构建了靶向人FS基因的shRNA干扰靶序列重组质粒(PLKO.1-FS-shRNA),转染后对人卵巢癌细胞系FS基因的表达具抑制效果,该实验为进一步研究FS基因的生物学功能奠定基础。Objective ： To construct the shRNA eukaryotic expression plasmid of Follistatin （FS） gene and preliminarily identi- fy its interference efficiency. Methods： According to the FS mRNA nucleotide sequence provided in GENEBANK database, the FS gene shRNA interference target sequence was selected by using the Ambion wed siRNA design software, then cloned in- to the linearized pLKO. 1 plasmid vector to construct the pLKO. 1 - FS - shRNA recombinant plasmids. After restriction en- zyme digestion and sequencing identification, the recombinant plasmids were transiently transfected into 3AO human ovarian cancer cell lines by lipofectamineTM 2000 method, then gene interference efficiency were identified by RT - PCR method. Re- suits： The recombinant shRNA sequence was confirmed by DNA sequencing, which was exactly the same as designed; RT- PCR results showed that the FS gene transcription within 3AO human ovarian cancer cells were suppressed after transiently transfected the pLKO. 1 - FS - shRNA recombinant plasmids, and the interference rate on mRNA expression of FS was 59%. Conclusion： The human FS gene pLKO. 1 - FS - shRNA recombinant plasmids was constructed successfully, which lays the foundation for further study of the biological function of FS gene.

关 键 词：卵泡抑素基因 RNA干扰 小发夹RNA 

分 类 号：R737.31[医药卫生—肿瘤]