真核基因重组Sox2及其在小鼠角膜基质细胞中的表达

发布时间：2019-07-27 11:37

【摘要】：目的基因重组构建具有穿膜活性的核转录因子Sox2真核表达系统,将其转染至小鼠角膜基质细胞,检测其在小鼠角膜基质细胞中的表达。方法利用重叠延伸PCR法重组目的基因Sox2-PTD构建质粒,测序检测重组DNA序列是否克隆正确。确定正确序列后将其转染至小鼠角膜基质细胞。倒置显微镜下观察细胞生长情况,转染后约10d免疫荧光化学鉴定其表达。结果成功构建质粒PCDNA3.1-Sox2-PTD;4个转录因子测序结果显示:Sox2基因测序结果与基因模板序列100%一致;成功转染至小鼠角膜基质细胞中;荧光显微镜下观察可见,Sox2-PTD在细胞浆和细胞核内均有表达,主要分布在细胞核内。结论成功构建核转录因子的真核表达系统,可转染至小鼠角膜基质细胞并引起细胞形态和表型的变化,为真核重组外源蛋白诱导角膜基质细胞转化为多能干细胞奠定基础。
[Abstract]:Objective to construct the eukaryotic expression system of nuclear transcription factor Sox2 with transmembrane activity and transfer it into mouse corneal stroma cells to detect its expression in mouse corneal stroma cells. Methods the recombinant target gene Sox2-PTD was constructed by overlapping extension PCR and sequenced to detect whether the recombinant DNA sequence was cloned correctly. The correct sequence was determined and transferred into mouse corneal stroma cells. The growth of the cells was observed under inverted microscope, and the expression of the cells was identified by immunofluorescence chemistry about 10 days after transfection. Results the sequencing results of plasmid PCDNA3.1-Sox2-PTD;4 transcription factors showed that the sequencing results of Sox2 gene were 100% consistent with those of gene template, and were successfully transformed into mouse corneal stroma cells. Under fluorescence microscope, Sox2-PTD was expressed in cytoplasm and nucleus, mainly distributed in nucleus. Conclusion the eukaryotic expression system of nuclear factor can be transformed into mouse corneal stroma cells and induce morphological and phenotypic changes, which lays a foundation for the transformation of corneal stroma cells into pluripotent stem cells induced by eukaryotic recombinant exogenous protein.
【作者单位】：郑州市第二人民医院眼科;暨南大学再生医学教育部重点实验室暨南大学医学院眼科研究所;
【基金】：国家自然科学基金资助(编号:30973244) 中央高校基本科研业务费专项资金资助(编号21609407) 郑州市医学博士创业基金(编号:121PPTGG495-7)~~
【分类号】：R341

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