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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 a small interfering RNA (siRNA) eukaryotic expression vector (pSUPER-HLA-G1) targeting human leukocyte antigen G1 (HLA-G1) and to detect its knockdown efficiency in the feeder cell line HTR-8 / SVneo . METHODS: The designed HLA-G1 siRNA oligodeoxynucleotide chain was linked with the eukaryotic expression vector pSUPER to construct a recombinant pSUPER-HLA-G1 eukaryotic expression vector and transfected into HTR-8 / SVneo cell line. The gene transcription of HLA-G1 in HTR-8 / SVneo cells was detected by RT-PCR after pSUPER-HLA-G1 plasmid transfection. The expression of HLA-G1 protein was detected by Western blotting. Results: The eukaryotic expression vector pSUPER-HLA-G1 was expressed in HTR-8 / SVneo cells. The inhibitory rates of HLA-G1 mRNA and its protein expression were 74.85 ± 7.43% and 71.07 ± 6.11%, respectively. CONCLUSION: The constructed human eukaryotic expression vector pSUPER-HLA-G1 for siRNA of human HLA-G1 effectively silences the gene expression and protein expression of HLA-G1 in HTR-8 / SVneo trophoblast cells. Point of gene research laid the foundation.