靶向AATF基因shRNA表达载体的构建及其稳定表达的白血病U937细胞系的建立

Construction of shRNA Expression Vector Targeting AATF and Establishment of Stably Transfected U937 Cells

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。

This study was aimed to construct the targeting AATF shRNA eukaryotic expression vector and establish the stably transfected U937 cell lines. The sequence of AATF mRNA was obtained from GenBank. After excluding ho meology, three plasmid expression vectors coding shRNA targeting 228 -249,303 - 324 and 443 -464 of AATF gene se quence were synthesized. Two terminals of shRNA carded BamH I and Hind 111 restriction sites. The selected nucleotides were cloned into the plasmid pSilencer 3.1-HI neo respectively, and the resultant recombinant plasmids were named as pSA-1 ,pSA-2 ,pSA-3. The sequences of the recombinant plasmids were identified by DNA sequencing. The recombinant plasmids were transfected into the cell line U937 by electroporation with NeonTM Transfection System. The transfected cells were persistently screened under G418 （500 mg/L）, and isolated with a limited dilution for 8 weeks. The inhibition of AATF mRNA and protein expression was respectively detected by RT-PCR and Western blot. The results indicated that RNAi eukaryotic expression vectors targeting AATF had correct reading frame and nucleotide sequence. Real-time PCR revealed that AATF shRNA effectively silenced mRNA expression of AATF. Western blot analysis found that AATF shRNA obviously suppressed protein expression of AATF（ P 〈0. 05 ）. It is concluded that the shRNA eukaryotic expression vector has been successfully constucted which can inhibit the expression of AATF, and the establishment of stably transfected U937 cell lines provide a original route for exploring the mechanism of AATF in human Leukemia further.