靶向人类白细胞抗原G1(HLA-G1)的siRNA真核表达载体的构建及表达  

Construction and Expression of the HLA-G1 siRNA Eukaryotic Expression Vector Eukaryotic Expression Vector

作　　者：李会俭[1] 刘学渊[1] 顾蔚蓉[1] 李笑天[1] 

机构地区：[1]复旦大学附属妇产科医院,上海200011

出　　处：《生殖与避孕》2011年第11期725-730,共6页Reproduction and Contraception

基　　金：上海市自然科学基金资助项目;项目号:10ZR1404900

摘　　要：目的:构建靶向人类白细胞抗原G1(HLA-G1)的小干扰RNA(siRNA)真核表达载体(pSUPER-HLA-G1),并检测其在滋养细胞系HTR-8/SVneo中的敲减效率。方法:将设计的HLA-G1 siRNA寡聚脱氧核苷酸链与真核表达载体pSUPER连接,构建重组pSUPER-HLA-G1真核表达载体,并用脂质体法将其转染入HTR-8/SVneo细胞系中。pSUPER-HLA-G1质粒转染后采用RT-PCR检测HLA-G1在HTR-8/SVneo细胞中的基因转录情况,Western blotting检测HLA-G1蛋白的表达情况。结果:构建的真核表达载体pSUPER-HLA-G1可在HTR-8/SVneo细胞中表达,HLA-G1 mRNA和其蛋白表达抑制率分别为74.85±7.43%和71.07±6.11%。结论:构建的人HLA-G1 siRNA蛋白真核表达载体pSUPER-HLA-G1有效地沉默了HLA-G1在HTR-8/SVneo滋养细胞中的基因表达及蛋白表达,为今后以HLA-G1为靶点的基因研究奠定了基础。Objective： To construct the HLA-G1 small intefering RNA（siRNA） eukaryotic expression vector（pSUPER-HLA-G1） and detect HLA-G1 expression in trophoblast cell line HTR-8/SVneo cells after transfection.Methods： The oligodeoxynucleotide chain of the HLA-G1 siRNA was connected with the eukaryotic expression vector pSUPER,then pSUPER-HLA-G1 eukaryotic expression vector was transfected into HTR-8/SVneo cells using lipofectamine transfection.The expression changes of HLA-G1 in mRNA and protein level in HTR-8/SVneo cells were respectively detected by RT-PCR and Western blotting.Results： HLA-G1 could be expressed by eukaryotic expression vector pSUPER-HLA-G1 in HTR-8/SVneo cells.The rates of inhibition HLA-G1 mRNA and its protein expression were 74.85 ±7.43% and 71.07 ± 6.11%,respectively.Conclusion： The expressions of HLA-G1 in mRNA and protein level were effectively silenced by pSUPER-HLA-G1 in HTR-8/SVneo cells.This may pave the road for function analysis of HLA-G1 gene in pre-eclampsia in the future.

关 键 词：人类白细胞抗原G1(HLA-G1) RNA干扰(RNAI) 真核表达载体 子痫前期 

分 类 号：R714[医药卫生—妇产科学]