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目的构建pre-miR-10a(miR-10a前体)重组腺病毒表达质粒,感染K562细胞,并检测其基因表达水平。方法化学合成pre-miR-10a基因序列,克隆至腺病毒穿梭质粒pAdTrack-CMV,将构建正确的重组腺病毒穿梭质粒pAdTrack-CMV pre-miR-10a与腺病毒骨架质粒pAdEasy-1共转化BJ5183感受态细胞,经同源重组获得重组腺病毒质粒pAd-pre-miR-10a,转染HEK293细胞,包装出重组腺病毒,经4轮扩增后检测病毒滴度。收集病毒后,以100 MOI感染K562细胞,RT-PCR法检测miR-10a基因的转录水平。结果重组腺病毒质粒pAd-pre-miR-10a经PacⅠ酶切,证明带有目的基因的重组腺病毒穿梭质粒已整合到腺病毒基因组中,4轮扩增后腺病毒滴度为1.5×1011pfu/ml,可高效感染K562细胞,感染效率达90%以上;重组腺病毒Ad-pre-miR-10a感染的K562细胞中miR-10a基因转录水平明显升高,过表达率为150.82%。结论已成功构建重组腺病毒表达质粒pAd-pre-miR-10a,并可在K562细胞中高效表达,为进一步从体内和体外水平研究其抗白血病效应奠定了基础。
Objective To construct the recombinant adenovirus expression vector of pre-miR-10a (miR-10a precursor), infect K562 cells and detect its gene expression. Methods The gene sequence of pre-miR-10a was chemically synthesized and cloned into shuttle adenovirus shuttle plasmid pAdTrack-CMV. The correct recombinant adenovirus shuttle plasmid pAdTrack-CMV pre-miR-10a and adenovirus backbone plasmid pAdEasy-1 were co-transformed into BJ5183 The recombinant adenovirus plasmid pAd-pre-miR-10a was obtained by homologous recombination and transfected into HEK293 cells. The recombinant adenovirus was packaged and tested for virus titer after 4 rounds of amplification. After collecting the virus, K562 cells were infected with 100 MOI, and the transcription level of miR-10a gene was detected by RT-PCR. Results Recombinant adenovirus plasmid pAd-pre-miR-10a was digested with PacⅠ, which proved that the recombinant adenovirus shuttle plasmid with the target gene was integrated into the adenovirus genome. The adenovirus titer after 4 rounds of amplification was 1.5 × 10 11 pfu / ml. The infection efficiency of K562 cells was over 90%. The transcription level of miR-10a gene in K562 cells infected with Ad-pre-miR-10a recombinant adenovirus was significantly increased, with an overexpression rate of 150.82%. Conclusion The recombinant adenovirus plasmid pAd-pre-miR-10a has been successfully constructed and expressed in K562 cells in vitro, which laid the foundation for the further study of its anti-leukemia effect in vivo and in vitro.