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目的:构建含大鼠ferroportin 1(FP1,膜铁转运蛋白1)基因和EGFP(绿色荧光蛋白)基因的过表达慢病毒载体并转染PC12细胞。方法:化学合成含有目的基因FP1的质粒,将酶切获得的目的基因连接入线性化慢病毒载体,构建重组质粒PGC-FU-FP1并进行测序鉴定;鉴定正确的阳性克隆采用Lipofectamine2000转染293T细胞,通过荧光显微镜和Western Blot检测FP1表达情况;在脂质体介导下将PGC-FU-FP1、pHelper1.0和pHelper2.0三质粒系统共转染入293T细胞,包装产生慢病毒,并通过实时荧光定量PCR法检测病毒滴度。以重组慢病毒载体质粒PGC-FU-FP1转染PC12细胞,荧光显微镜下观察EGFP的表达,实时荧光定量PCR和Western Blot法分别检测FP1mRNA和蛋白表达情况。结果:FP1基因序列经测序后与GeneBank报道的序列完全一致;重组质粒PGC-FU-FP1中携带有正确的FP1基因并能在293T细胞中表达;病毒滴度为2×108TU/ml。荧光显微镜、实时荧光定量PCR和Western Blot法证实目的基因FP1能被重组慢病毒高效导入PC12细胞并得到过表达。结论:成功构建携带FP1基因的慢病毒载体,并获得FP1基因修饰的PC12基因工程细胞。为进一步研究FP1在帕金森病中的作用及基因治疗奠定基础。
OBJECTIVE: To construct an over-expressing lentiviral vector containing rat ferroportin 1 (FP1, membrane iron transporter 1) gene and EGFP (green fluorescent protein) gene and transfect it into PC12 cells. Methods: The plasmid containing the target gene FP1 was chemically synthesized. The target gene was ligated into the lentiviral vector and the recombinant plasmid PGC-FU-FP1 was constructed and sequenced. The correct positive clones were identified and transfected into 293T cells by Lipofectamine 2000 The expression of FP1 was detected by fluorescence microscopy and Western Blot. The 293T cells were co-transfected with 293T cells under the liposome-mediated three-plasmid system of PGC-FU-FP1, pHelper1.0 and pHelper2.0. Real-time fluorescence quantitative PCR detection of virus titer. PC12 cells were transfected with the recombinant lentiviral vector PGC-FU-FP1. The expression of EGFP was observed under a fluorescence microscope. The expression of FP1 mRNA and protein was detected by real-time fluorescence quantitative PCR and Western Blot. Results: The sequence of FP1 gene was identical to the sequence reported in GeneBank. The recombinant plasmid PGC-FU-FP1 contained the correct FP1 gene and expressed in 293T cells. The virus titer was 2 × 108 TU / ml. Fluorescence microscopy, real-time fluorescence quantitative PCR and Western Blot confirmed that the target gene FP1 was efficiently introduced into PC12 cells by recombinant lentivirus and was overexpressed. Conclusion: The lentiviral vector carrying FP1 gene was successfully constructed and PC12 genetically engineered cells with FP1 gene modification were obtained. To further study the role of FP1 in Parkinson’s disease and gene therapy.