HLA-G基因siRNA真核表达质粒的构建及其对转染JEG-3细胞株HLA-G表达的抑制作用  

Construction of eukaryotic expression plasmid expressing siRNA targeting HLA-G gene and its specific downregulation of HLA-G expression in transfected JEG-3 cells

作　　者：陈建辉[1] 姚元庆[1] 侯开波[1] 王晓蓉[2] 雷迎峰[3] 尹文[3] 

机构地区：[1]第四军医大学唐都医院妇产科 [2]武警黑龙江省总队医院妇产科,哈尔滨市150076 [3]第四军医大学基础部微生物学教研室,西安市710032

出　　处：《实用医学杂志》2007年第15期2295-2298,共4页The Journal of Practical Medicine

基　　金：国家自然科学基金资助项目(编号:30471812)

摘　　要：目的:构建针对人类白细胞抗原-G基因(human leukocyte antigen-G,HLA-G)基因siRNA表达质粒,观察其对转染JEG-3细胞的HLA-G基因表达的抑制作用。方法:选择RNA干涉靶序列,体外合成两段互补的寡核苷酸,通过与线性化pSuppressor-U6-neo连接、转化大肠杆菌,扩增、纯化得到所需质粒,通过琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列。将重组真核表达质粒pSuppressor-U6-neo-HLA-G用脂质体法转染JEG-3绒癌细胞株。应用RT-PCR、Western-blot方法检测转染的JEG-3细胞中HLA-G基因的表达水平。结果:双酶切鉴定及测序图显示,插入的寡核苷酸序列与设计的序列完全相符。RT-PCR和Western-blot结果均表明瞬时转染重组pSuppressor-U6-neo-HLA-G质粒明显抑制了JEG-3细胞中HLA-G基因的表达。结论:本试验成功构建了针对HLA-G基因的siRNAs表达质粒pSuppressor-U6-neo-HLA-G,瞬时转染JEG-3细胞后可以明显抑制HLA-G基因的表达。Objective To construct siRNA expressing plasmid aimed at HLA-G gene and investigate its inhibitory effect on the expression of HLA-G in JEG-3 cells. Methods A target sequence in the middle of HLA-G gene was selected. Two complementary oligonucleotides were synthesized in vitro and then ligated via linearized pSuppressorU6-neo. The plasmid was transformed into E.coli DHSa to amplify and purified. The purified plasmid was identified by gel electrophoresis and sequencing. The resultant plasmid pSuppressor-U6-neo-HLA-G was transfected into JEG-3 cells with lipofectamine 2000. The non-transfected cells, cells transfected by pSuppressor-U6-neo, and non-specific siRNA transtrected cells were used as controls. The inhibitory effects of HLA-G mRNA and protein expression were detected by semi-quantitative RT-PCR and Western-blot, respectively. Result The gel electrophoresis and sequencing showed that the inserted sequence was identical to the one we designed and no aberrations such as mutation ,deletion or insertion occurred. Conclusions The plasmid pSuppressor-U6-neo-HLA-G which can express siRNA aimed at HLA-G gene has been constructed successfully. Transient transfection can significantly decrease the level of HLA-G expression in transfected JEG-3 cells.

关 键 词：HLA抗原 RNA 小分子干扰 转染 JEG-3细胞 

分 类 号：R392[医药卫生—免疫学]