Pre-miR-10a重组腺病毒质粒的构建及其在K562细胞中的表达  

Construction of recombinant adenovirus vector for pre-miR-10a and its expression in K562 cells

作　　者：齐杰玉[1] 陶崑[1] 王灿蔚[1] 李秋红[2] 邓一平[3] 

机构地区：[1]重庆医科大学基础医学院免疫学教研室重庆医科大学分子医学与肿瘤研究中心,重庆400016 [2]重庆市妇幼保健院检验科,重庆400013 [3]重庆医科大学实验中心,重庆400016

出　　处：《中国生物制品学杂志》2013年第5期657-660,共4页Chinese Journal of Biologicals

摘　　要：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 vector for pre-miR-10a and determine its expression level in K562 cells.Methods The gene sequence of pre-miR-10a was synthesized chemically and cloned into adenovirus shuttle vector pAdTrack-CMV.The constructed recombinant adenovirus shuttle plasmid pAdTrack-CMV-pre-miR-10a co-transfected to competent BJ5183 cells with adenovirus backbone plasmid pAdEasy-1.Recombinant adenovirus plasmid pAdpre-miR-10a obtained by homologous recombination was transfected to HEK 293 cells for packaging.The obtained recombinant adenovirus was determined for titer after four cycles of propagation.K562 cells were infected with the harvested recombinant adenovirus at a MOI of 100,and determined for transcription level of miR-10a gene by RT-PCR.Results Digestion of recombinant adenovirus plasmid pAd-pre-miR-10a with Pac Ⅰ proved that recombinant adenovirus shuttle plasmid carrying target gene was integrated to the genome of adenovirus.The titer of recombinant adenovirus after four cycles of propagation was 1.5×10 11 pfu/ml,of which the infection efficacy to K562 cells was more than 90%.The transcription level of miR-10a gene in K562 cells infected with recombinant adenovirus Ad-pre-miR-10a increased significantly,with an over-expression rate of 150.82%.Conclusion Recombinant adenovirus expression vector pAd-pre-miR10a was successfully constructed and high expressed in K562 cells,which laid a foundation of further study on the in vitro and in vivo anti-leukemia function of pre-miR-10a.

关 键 词：pre-miR-10a 腺病毒科 K562细胞 基因表达 

分 类 号：Q782[生物学—分子生物学] Q786