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目的构建携带PML(NLS-)基因的重组腺病毒,并观察其对白血病K562细胞增殖的影响。方法以质粒pCMV-HA-PML(NLS-)为模板,PCR扩增PML(NLS-)基因,与穿梭质粒pAdTrace-TO4连接,构建重组穿梭载体pAdTrace-TO4-PML(NLS-),经PmeⅠ酶切线性化后,转化入感受态BJ5183细菌,获得重组腺病毒质粒pAd-PML(NLS-),经PacⅠ酶切后,转染AD293细胞,获得重组腺病毒Ad-PML(NLS-),经4轮扩增后,检测其滴度。采用RT-PCR法和Western blot法分别检测重组腺病毒中PML(NLS-)基因mRNA的转录水平和蛋白的表达水平;将重组腺病毒感染K562细胞,MTT法检测其对细胞增殖活力的影响。结果重组腺病毒质粒pAd-PML(NLS-)经PCR和单酶切鉴定,证明构建正确;经4轮扩增,重组腺病毒的滴度为1×1010pfu/ml;Ad-PML(NLS-)携带的PML(NLS-)能够在AD293细胞中稳定表达;与未感染组和空载病毒感染组相比,重组腺病毒Ad-PML(NLS-)感染的K562细胞的增殖活力明显增强(P<0.05)。结论成功构建了重组腺病毒Ad-PML(NLS-),其可促进K562细胞的增殖,表明PML(NLS-)可能具有促癌基因的作用。
Objective To construct a recombinant adenovirus carrying PML (NLS-) gene and observe its effect on the proliferation of K562 leukemia cells. METHODS: PML (NLS-) gene was amplified by PCR using plasmid pCMV-HA-PML (NLS-) as a template and ligated with shuttle plasmid pAdTrace-TO4 to construct recombinant shuttle vector pAdTrace-TO4-PML The recombinant adenovirus plasmid pAd-PML (NLS-) was transformed into competent BJ5183 bacteria. After digestion with Pac I, the recombinant adenovirus was transfected into AD293 cells to obtain recombinant adenovirus Ad-PML (NLS-) After rounds of amplification, the titers were tested. The transcription level and protein expression of PML (NLS-) mRNA in recombinant adenovirus were detected by RT-PCR and Western blot, respectively. K562 cells were infected with recombinant adenovirus and the effect on cell proliferation was tested by MTT assay. Results The recombinant adenovirus plasmid pAd-PML (NLS-) was confirmed by PCR and single enzyme digestion. The recombinant adenovirus plasmid pAd-PML (NLS-) was constructed correctly. After four rounds of amplification, the recombinant adenovirus titer was 1 × 1010pfu / PML (NLS-) carried by the recombinant adenovirus could stably express in AD293 cells. The proliferation activity of K562 cells infected with recombinant adenovirus Ad-PML (NLS-) was significantly higher than that of uninfected and uninfected (P < 0.05). Conclusion The recombinant adenovirus Ad-PML (NLS-) was successfully constructed, which can promote the proliferation of K562 cells, indicating that PML (NLS-) may have the role of oncogene.