hsa-miR-150慢病毒表达质粒的构建及鉴定  

Construction and identification of lentiviral expression vector hsa-miR-150

作　　者：张俊[1] 张涛[2] 王勃[1] 李少林[1] 

机构地区：[1]重庆医科大学放射医学教研室,重庆400016 [2]重庆医科大学附属第一医院肿瘤科,重庆400016

出　　处：《中国生物制品学杂志》2012年第2期140-143,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30970843)

摘　　要：目的构建hsa-miR-150慢病毒表达质粒,并鉴定其感染肝癌细胞系Hep3B后miR-150的表达水平。方法根据hsa-miR-150序列化学合成hsa-miR-150基因片段,克隆至慢病毒载体pGIPZ上,构建重组慢病毒表达质粒pGIPZ-hsa-miR-150,转染包装细胞293T进行包装,检测病毒滴度。收获并浓缩重组慢病毒颗粒,稳定转染Hep3B细胞,流式细胞术检测GFP的表达率;荧光定量PCR检测miR-150的表达水平。结果重组慢病毒表达质粒pGIPZ-hsa-miR-150经菌落PCR及测序证实构建正确;重组慢病毒的滴度约为5.7×108 TU/ml;稳定转染Hep3B细胞后,GFP的表达率为(97.78±1.14)%,重组慢病毒转染组miR-150的表达水平较空白对照组增加约6倍(P<0.05)。结论成功构建了hsa-miR-150慢病毒表达质粒,其在Hep3B细胞中可高效表达,为进一步研究miR-150的生物学功能奠定了基础。Objective To construct lentiviral expression vector hsa-miR-150 and determine the expression of miR-150 in infected hepatocellular carcinoma cell line Hep3B. Methods The hsa-miR-150 gene fragment was synthesized chemically and cloned into lentiviral vector pGIPZ. The constructed recombinant plasmid pGIPZ-hsa-miR-150 was transfected to packaging cell line 293T, and the packaged virus was determined for titer. Recombinant lentivirus particles were harvested and concentrated, then stably transfected to Hep3B cells and determined for expression rate of GFP by flow cytometry, and for expression level of miR-150 by fluorescent quantitative PCR. Results PCR amplification of bacterial colony and sequencing proved that recombinant lentiviral expression vector pGIPZ-hsa-miR-150 was constructed correctly. The titer of recombinant lentivirus was 5. 7×l0^[8] / ml. The expression rate of GFP in Hep3B cells stably transfected with pGIPZ-hsa-miR-150 was （97.78 ＋ 1. 14）%, while the expression level of miR-150 increased by 6-folds as compared with those in blank control group （P 〈 0. 05）. Conclusion The lentiviral expression vector hsa-miR-150 was constructed successfully and highly expressed in Hep3B, which laid a foundation of further study on biological function of miR-150.

关 键 词：微RNAS hsa-miR-150 慢病毒载体 肝肿瘤 

分 类 号：R318[医药卫生—生物医学工程] Q782[医药卫生—基础医学]