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目的:构建过氧化物酶蛋白1(PRDX 1)的真核表达载体,观察其在Hela细胞内表达及定位。方法:提取BALB/c小鼠肝脏组织总RNA,通过RT-PCR方法扩增得到小鼠PRDX 1编码序列,酶切后克隆至pcDNA3-myc载体。重组质粒通过PCR、酶切、测序证明构建正确后经脂质体转染Hela细胞,然后利用Western blot和荧光显微镜技术观察该融合蛋白在细胞内表达及定位。结果:经鉴定证明重组质粒构建正确;Western blot实验显示,该质粒能够在Hela细胞中特异表达;免疫荧光试验显示,蛋白产物分布在胞浆和胞核,证明该蛋白在细胞内高表达。结论:成功构建带有myc标签的PRDX 1真核表达载体,该质粒能够在哺乳细胞中特异表达并且外源性PRDX 1蛋白分布在Hela细胞胞浆胞核内,为深入研究PRDX 1蛋白在细胞内相关生物学研究奠定了基础。
OBJECTIVE: To construct the eukaryotic expression vector of peroxidase protein 1 (PRDX 1) and to observe its expression and localization in Hela cells. Methods: The total RNA of BALB / c mice liver tissue was extracted. The coding sequence of PRDX 1 was amplified by RT-PCR and cloned into pcDNA3-myc vector. Recombinant plasmids were transfected into Hela cells by lipofectamine after they were constructed correctly by PCR, restriction enzyme digestion and sequencing. Then the expression and localization of the fusion protein in Hela cells were observed by Western blot and fluorescence microscopy. Results: The recombinant plasmids were constructed correctly. Western blot showed that the plasmids were specifically expressed in Hela cells. Immunofluorescence assay showed that the protein products were distributed in the cytoplasm and nucleus, indicating that the protein was highly expressed in the cells. CONCLUSION: The PRDX 1 eukaryotic expression vector with myc tag was successfully constructed. The plasmid can be specifically expressed in mammalian cells and the exogenous PRDX 1 protein is distributed in the cytoplasmic nucleus of Hela cells. In order to further study the expression of PRDX 1 in cells The relevant biological research laid the foundation.