重组腺病毒Ad-PML(NLS)-的构建及鉴定  

Construction and identification of recombinant adenovirus Ad-PML(NLS^-)

作　　者：高远梅[1,2] 刘北忠[1,2] 张曦[2] 胡秀秀[2] 钟梁[2] 

机构地区：[1]重庆医科大学附属永川医院中心实验室,重庆402160 [2]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2013年第7期906-911,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81171658);重庆市自然科学基金计划重点项目(2011BA5037)

摘　　要：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 recombinant adenovirus carrying PML（NLS-） gene and observe its＇ effect on the proliferation of leukemia K562 cells.Methods PML（NLS-）gene was amplified by PCR using plasmid pCMV-HA-PML（NLS-） as a template and inserted into shuttle plasmid pAdTrace-TO4.The constructed recombinant shuttle plasmid pAdTrace-TO4-PML（NLS-） was digested with PmeⅠ and transformed to competent BJ5183.The obtained recombinant adenovirus plasmid pAd-PML（NLS-） was digested with PacⅠ and transfected to AD293 cells for packaging.The obtained recombinant adenovirus Ad-PML（NLS-） was determined for titer after four cycles of amplification,and for PML（NLS-） mRNA transcription and protein expression by RT-PCR and Western blot respectively.K562 cells were infected with the recombinant adenovirus and determined for proliferation activity by MTT method.Results Recombinant adenovirus plasmid pAd-PML（NLS-） was constructed correctly as proved by PCR and restriction analysis.The titer of recombinant adenovirus after four cycles of amplification was 1 × 1010 PFU / ml.The PML（NLS-） gene in Ad-PML（NLS-） was expressed stably in AD293 cells.The proliferation activity of K562 cells infected with Ad-PML（NLS-） increased significantly as compared with those uninfected and those infected with Ad-KZ（P 0.05）.Conclusion Recombinant adenovirus Ad-PML（NLS-） was constructed successfully,which promoted the proliferation of K562 cells.It indicated the possible promoting effect of PML（NLS-） on oncogene.

关 键 词：PML(NLS-)基因 重组腺病毒 K562细胞 细胞增殖 

分 类 号：Q782[生物学—分子生物学] R733.72[医药卫生—肿瘤]