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本研究旨在构建靶向AATF基因的短发夹RNA(shRNA)真核表达载体,获得干扰质粒稳定转染的人白血病U937细胞系。根据GenBank数据库提供的AATF mRNA序列,以228～249、303～324、443～464位序列作为RNA干扰位点,排除同源可能,合成3对互补并编码mRNA基因的特异性shRNA序列的寡核苷酸,克隆至经BamHⅠ、HindⅢ双酶切线性化的pSilencer 3.1人H1启动子干扰载体中,利用测序鉴定所构建的重组载体是否正确,经电穿孔将3个重组载体(pSA-1、pSA-2、pSA-3)转染人白血病U937细胞系,G418(500 mg/L)有限稀释法持续筛选8周后,扩增获得稳定表达的细胞系;RT-PCR、Western blot分别检测稳定转染细胞系的AATF mRNA和AATF蛋白表达。结果表明,AATF干扰序列及读码框完全正确。稳定转染细胞AATF mRNA表达下调,AATF蛋白表达量下降(P<0.05)。结论:成功地构建AATF shRNA真核表达载体及AATF表达稳定抑制的白血病U937细胞系,为以AATF基因为靶点的人白血病进一步研究奠定了实验基础。
The purpose of this study was to construct a short hairpin RNA (shRNA) eukaryotic expression vector targeting AATF gene and to obtain a human leukemia U937 cell line stably transfected into the plasmid. According to the AATF mRNA sequence provided by the GenBank database, 228 pairs of amino acid sequences of 249,303 ~ 324,443 ~ 464 were used as RNA interference sites to eliminate the possibility of homology and to synthesize three pairs of oligonucleotides complementary to the specific shRNA sequence encoding the mRNA gene (PSA-1, pSA-1, pSA-1, pSA-1) were digested by restriction endonucleases BamHⅠand HindⅢ, and sequenced to confirm whether the constructed recombinant vector was correct. -2, pSA-3) were transfected into U937 human leukemia cell line. After limited screening by G418 (500 mg / L) for 8 weeks, the stable cell lines were obtained by RT-PCR and Western blot. AATF mRNA and AATF protein expression of the stained cell lines. The results showed that the AATF interference sequence and the reading frame were completely correct. AATF mRNA expression was down-regulated in stable transfected cells, and AATF protein expression was decreased (P <0.05). Conclusion: The eukaryotic expression vector of AATF shRNA and the leukemia U937 cell line with stable expression of AATF were successfully constructed, which laid the experimental foundation for the further study of human leukemia targeting AATF gene.