摘要
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 novel isoform of PIK3IP1 and analyze their effects on cell viability, subcellular localization, and expression profile in cell lines. Methods： RT-PCR was used to clone PIK3IP1 and its novel splicing isoform PIK3IPI-vl from multi-tissue cDNA pool. By cell-based assays, we studied how PIK3IP1 and PIK3IPI-vl affected the activity of Renila luciferase and morphological change in the HEK 293T cells. Furthermore, flow cytometry experiment was used to validate that overexpression of both splicing isoforms could induce cell apoptosis. Bioinformaties analysis was used to identify structural characteristics of these two splicing isoforms. By fluorescence microscopy assay, we identified their subcellular localization. RT-PCR was used to detect the expression of PIK3IP1 in the cell lines. Results： PIK3IP1 and its novel splicing isoform PIK3IPI-vl were cloned and constructed into the peDNA-and pEGFP-expression plasmids. They both had signal peptide and transmembrane domain. Nevertheless, localization on cell membrane and lowly expressed in many cell lines. Conclusion： PIK3IPI-vl is a novel splicing isoform of PIK3IP1. Both of them are located on cell membrane and can induce cell apoptosis. KE
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2008年第6期572-577,共6页
Journal of Peking University:Health Sciences
基金
国家高技术研究发展计划专项经费资助(2006AA02A305)~~
