人肺癌转移相关蛋白1重组慢病毒载体的构建和病毒包装及其鉴定  

Construction of human lung cancer metastasis related protein 1 recombinant lentiviral vector and virus packaging and identification

作　　者：李彦芹[1] 于玲[1] 梁媛[1] 于清扬[1] 刘扬[1] 李春笋[1] 

机构地区：[1]解放军总医院呼吸科,北京100853

出　　处：《中国医药》2013年第5期615-617,共3页China Medicine

基　　金：基金项目：国家自然科学基金项目（30370616/H1615）;中国博士后科学基金（20090461438）

摘　　要：目的构建人肺癌转移相关蛋白1（LCMR1）重组慢病毒载体，并包装慢病毒颗粒，为进一步研究该基因在人肺癌发生发展中的功能提供实验基础。方法将人肺癌转移相关基因LCMR1扩增后，酶切连接构建人pLVX—IRES—Neo病毒载体，转化DH5a大肠杆菌，挑取阳性克隆测序鉴定，将重组质粒与慢病毒系统一起转染Lenti—X293T病毒包装细胞，并将收集的病毒感染肺癌细胞系95C，即时定量聚合酶链反应（q—PCR）及蛋白质印迹法验证其表达情况。结果DNA测序结果证实成功构建人LCMR1重组慢病毒载体，包装后的病毒浓缩液浓度可达2．5×10^8IFU／ml以上。包装后的病毒颗粒感染95C细胞系，LCMR1的mRNA水平提高9．98倍（P〈0．05），蛋白水平的表达也明显增强。结论本研究成功构建了pLVX—IRES—LCMR1-Neo慢病毒载体，获得的慢病毒颗粒有效感染肺癌细胞系95C，为研究该基因在肺癌中的功能建立了模型。Objective To construct a lentiviral expressing vector carrying human lung cancer metastasis re- lated protein 1 （LCMR1） gene and then the overexpression in lung cancer 95C cell line was tested in order to pro- vide an experimental foundation for the function of LCMR1 on lung cancer. Methods LCMR1 gene cDNA was am- plicated by polymerase chain reaction （PCR） and cloned into the pLVX-IRES-Neo vector. Reconstructed pLVX- IRES-Neo-LCMR1 plasmid was identified by electromigratory analysis, colony PCR and sequencing analysis. The vi- ral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells. After infection of 95C cells with these lentiviruses, the expression of LCMR1 was detected by q-PCR and Western blot. Results LC- MR1 gene was constructed into the pLVX-IRES-Neo vector successfully. Enzyme digestion got correct length of LC- MR1 gene, DNA sequencing analysis confirmed that LCMR1 gene sequence was exactly the same with that reported by GenBank. There were significant increasing expressions of LCMR1 mRNA and protein in the 95C cells after transfection（P 〈 0.05 ）. Conclusions All the recombinant plasmids can be confirmed by sequencing. The 95C cells infected with lentiviral vector carrying full length LCMR1 gene express high-level LCMR1 successfully.

关 键 词：肺癌转移相关蛋白1 慢病毒 载体构建 肺癌 

分 类 号：R734.2[医药卫生—肿瘤]