磁共振报告基因magA的慢病毒载体质粒构建及体外表达  

Construction of recombinant lentivirus expression vector carrying MagA and its in vitro expression

作　　者：秦勇[1,2] 蔡金华[1] 郑鹤琳[1] 刘官信[2] 王世一[2] 刘波[1] 

机构地区：[1]重庆医科大学附属儿童医院放射科,重庆400014 [2]重庆医科大学附属儿童医院儿童发育疾病研究省部共建教育部重点实验室,儿科学重庆市重点实验室,重庆市儿童发育重大疾病诊治与预防国际科技合作基地,重庆400014

出　　处：《第三军医大学学报》2011年第13期1346-1349,共4页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC2009JJ0305)~~

摘　　要：目的构建携带磁共振报告基因magA的慢病毒载体质粒,初步检测其体外转铁效应。方法采用人工DNA合成技术合成目的基因magA,DNA重组技术将magA基因连接入慢病毒表达载体质粒pLenti-EGFP,酶切及DNA测序鉴定重组质粒pLenti-EGFP/magA的准确性。包装、包膜及重组质粒共转染293FT细胞包装慢病毒,收集包被magA基因的慢病毒上清并感染293T靶细胞,细胞培养基内加入500μmol/L枸橼酸铁连续4次传代,荧光显微镜观察并提取细胞行台盼蓝排除实验和普鲁士蓝染色,同时设立对照组行同样方法实验。结果酶切和DNA测序分析证实magA基因准确克隆入慢病毒表达载体设计位点,合成目的基因序列与GenBank中magA序列完全一致。荧光显微镜下观察包装细胞293FT细胞质内有大量绿色荧光表达,病毒滴度达到108 Tu/μl。慢病毒感染293T靶细胞后,细胞内稳定表达EGFP,且效率>80%。实验组及对照组台盼蓝拒染率分别为(92.80±2.65)、(93.50±1.29),2组拒染率差异无统计学意义(P>0.05)。普鲁士蓝染色发现实验组细胞内有大量蓝染铁颗粒形成,对照组呈阴性。结论 成功构建了携带磁共振报告基因magA的慢病毒表达载体,证实了magA基因在哺乳动物293T靶细胞内的转铁作用。Objective To construct a recombinant lentivirus expression vector encoding magA,a new MRI reporter gene,and to determine the iron-transporting effect of in vitro expressed magA.Methods The gene sequence of magA was synthesized by synthetic DNA technology,and then inserted into the lentivirus expression vector pLenti-EGFP by recombinant DNA technique.The recombinant lentivirus expression vector pLenti-EGFP/magA was verified by enzyme digestion and DNA sequencing.Then the recombinant lentivirus was packaged in the 293FT cells by co-transfecting with packaging plasmid,envelope plasmid,and lentiviral vector plasmid.The 293T cells were transfected with the packaged lentivirus particles,and then cultured in the medium containing 500 μmol/L ferric citrate.Prussian blue staining was performed to detect the iron particles in the 293T cells.Viability was assessed via Trypan blue exclusion testing.Results The enzyme digestion and DNA sequencing confirmed that the sequence of PCR-amplified magA was consistent with that in Genbank.The magA were cloned into pLenti-EGFP in a right direction.The three plasmids were transferred into 293T cells,which emitted green fluorescence under fluorescence microscope.The titer of resulting virus reached 108 TU/μl and infected 293T cells successfully with an efficiency of more than 80%.Prussian blue staining revealed numerous iron particles in the cytoplasm of 293T cells.Cell viability was〉 90% for all cell with no noteworthy difference between the control group and the experimental group.Conclusion The recombinant lentivirus expression vector of magA is successfully constructed and the iron transporting effect of magA in mammal 293T cells is verified,which provides a basis for further exploring MRI tracking in cells in vivo by using magA as a reporter gene.

关 键 词：magA 报告基因 磁共振成像 慢病毒载体 

分 类 号：R394-33[医药卫生—医学遗传学] R394.2[医药卫生—基础医学]