【摘要】 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。更多还原

【Abstract】 Objective: To construct a recombinant lentiviral vector carrying rat ferroportin 1(FP1) gene and EGFP gene and investigate the expression of FP1 in transfected PC12 cells.Methods: PGC-FU-FP1 plasmid was constructed by double restriction enzyme digestion and ligation,Then the plasmid was transformed into E.coli competent cells.The positive clones were identified by PCR and the sequence of insert was verified by DNA sequencing.The PGC-FU-FP1 DNA was extracted from positive clone and transfected into human embryonic kidney(HEK)-293T cells.The fluorescent microscopy and Western Blot were used to detect the expression of FP1 in 293T cells.Purified PGC-FU-FP1,pHelper1.0 and pHelper2.0 plasmids were cotransfected into 293T cells by Lipofectamine2000 to produce lentivirus,and the virus titer was determined by real-time fluorescence quantitative PCR(RT-qPCR) analysis.Finally,the recombinant lentiviral vector PGC-FU-FP1 was transfected into PC12 cells and overexpression of FP1 was examined by fluorescent microscopy,RT-qPCR and Western Blot analysis.Results: The sequence of FP1 gene in recombinant PGC-FU-FP1 was consistent with that reported in the GeneBank.The virus titer was 2×108 TU/ml.FP1 gene could be efficiently transfected into PC12 cells by lentiviral vector and achieved stable over-expression in PC12 cells.Conclusions: We successfully constructed FP1 expressing lentiviral vector and transfected it into PC12 cells to obtain the genetically modified PC12 cells.This study provided a novel insight into the role of FP1 and gene therapy in Parkinson’s disease.更多还原