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目的:发现和克隆人类基因PIK3IP1的新变异体并鉴定其功能,研究其对细胞活力的影响和亚细胞定位,分析PIK3IP1在细胞系中的表达情况。方法:利用GenBank中的数据,从人的混合组织cDNA中通过PCR克隆PIK3IP1和它的新剪切体PIK3IP1-v1,运用生物信息学分析其结构特性。经萤光素酶活性检测、细胞形态观察和流式细胞检测研究PIK3IP1和PIK3IP1-v1对细胞存活力的影响,使用荧光显微镜观察其亚细胞定位。最后通过RT-PCR分析PIK3IP1在细胞系中的表达情况。结果:获得PIK3IP1和新剪切体PIK3IP1-v1的真核表达质粒和融合绿色荧光蛋白表达质粒。生物信息学分析显示,PIK3IP1和PIK3IP1-v1均有信号肽并包含跨膜结构域,但后者胞外缺失一个Kringle结构域。功能分析发现,PIK3IP1和PIK3IP1-v1均可诱导细胞凋亡。荧光显微观察证实,PIK3IP1的两种剪切体均定位于细胞膜。RT-PCR证明PIK3IP1在多种细胞中转录水平较低。结论:新剪切体PIK3IP1-v1和PIK3IP1均定位于细胞膜并诱导细胞凋亡,而且在多种细胞中低水平转录。
OBJECTIVE: To find and clone a new variant of human PIK3IP1 gene and identify its function. To investigate its effect on cell viability and subcellular location, analyze the expression of PIK3IP1 in cell lines. Methods: Using the data in GenBank, PIK3IP1 and its new PIK3IP1-v1 were cloned by PCR from human mixed cDNA. The structural characteristics were analyzed by bioinformatics. The effects of PIK3IP1 and PIK3IP1-v1 on cell viability were examined by luciferase activity assay, cell morphology observation and flow cytometry. The subcellular localization was observed by fluorescence microscopy. Finally, the expression of PIK3IP1 in cell lines was analyzed by RT-PCR. Results: Eukaryotic expression plasmid and fusion green fluorescent protein expression plasmid of PIK3IP1 and newly-spliced PIK3IP1-v1 were obtained. Bioinformatics analysis showed that both PIK3IP1 and PIK3IP1-v1 had signal peptide and contained a transmembrane domain, but the latter had a Kringle domain extracellularly. Functional analysis found that PIK3IP1 and PIK3IP1-v1 can induce apoptosis. Fluorescence microscopy confirmed that both the PIK3IP1 spliceosomes localized to the cell membrane. RT-PCR proved that PIK3IP1 transcripts were lower in many kinds of cells. Conclusion: The new-cut PIK3IP1-v1 and PIK3IP1 are both located in the cell membrane and induce apoptosis, but also transcribed at a low level in many kinds of cells.