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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 lentiviral plasmid carrying the magnetic resonance reporter gene magA and to detect the iron transfer effect in vitro. Methods The target gene magA was synthesized by artificial DNA synthesis. The recombinant plasmids pLenti-EGFP / magA were identified by ligating the magA gene into lentiviral vector pLenti-EGFP. 293FT cells were co-transfected with the lentivirus by packaging, envelope and recombinant plasmids. The lentivirus coated with magA gene was collected and infected with 293T target cells. 500μmol / L iron citrate was added into the cell culture media for 4 consecutive passages. Fluorescence The cells were observed under microscope and were subjected to trypan blue exclusion assay and Prussian blue staining. At the same time, the control group was set up to conduct the same experiment. Results Enzyme digestion and DNA sequencing confirmed that magA gene was correctly cloned into the lentiviral vector. The sequence of the target gene was exactly the same as the sequence of magA in GenBank. Under fluorescent microscope, a large amount of green fluorescence was expressed in the 293FT cytoplasm of the packaged cells, and the virus titer reached 108 Tu / μl. Lentiviral 293T target cells infected with stable expression of intracellular EGFP, and the efficiency of> 80%. The rates of trypan blue exclusion in the experimental group and the control group were (92.80 ± 2.65) and (93.50 ± 1.29), respectively. There was no significant difference between the two groups (P> 0.05). Prussian blue staining found that a large number of blue-stained iron particles in the experimental group cells, the control group was negative. Conclusion The lentiviral expression vector carrying magnetic resonance reporter gene magA was successfully constructed, which confirmed the transfer of magA gene in mammalian 293T target cells.