人组氨酸磷酸酶蛋白PHPT1的细胞定位及其对细胞层状伪足形成的影响  

The Location of GFP Fused PHPT1 and Its Effect on Lamellipodia Formation

作　　者：徐安健[1] 李艳萌[1] 李斯文[1] 乌姗娜[2] 张蓓[1] 黄坚[1] 

机构地区：[1]首都医科大学附属北京友谊医院科研实验中心,北京100875 [2]首都医科大学附属北京友谊医院临床检验中心,北京100875

出　　处：《中国生物工程杂志》2017年第6期17-21,共5页China Biotechnology

基　　金：北京优秀人才培养项目资助项目(2016000021469G227)

摘　　要：目的:获取人组氨酸磷酸酶蛋白PHPT1基因,并构建其C端GFP融合的真核表达载体,通过瞬时转染观察融合蛋白在细胞内的表达和定位,并研究其定位与细胞层状伪足形成的关系。方法:以人宫颈癌细胞株HeLa cDNA为模板,PCR扩增PHPT1的全长编码基因,克隆到pEGFPN2载体中,构建pEGFP-N2-PHPT1真核表达载体,利用脂质体将构建的载体转染到HeLa细胞中,用激光共聚焦扫描显微镜观察C端GFP连接的PHPT1的细胞定位,并进一步探讨其定位与细胞层状伪足形成的关系。结果:成功构建了PHPT1的GFP融合表达载体pEGFP-N2-PHPT1,并在He La细胞中检测到了融合蛋白的表达,发现其定位与细胞层状伪足的形成密切相关,并可直接影响细胞的运动能力。结论:GFP融合形式表达的PHPT1蛋白在细胞质和细胞核中均有表达,并且定位于细胞层状伪足的前沿,影响细胞层状伪足的形成,从而影响细胞运动。Objective： To amplify human PHFF1 gene, and construct the C-terminal GFP fused vectors, investigate the expression and location of the fusion proteins in cells, as well as the relationship with lamellipodia. Methods： The cDNA sequence of PHP1 gene was amplified and subcloned into pEGFP-N2 vectors. Transfecting of those vectors into HeLa cells to investigating the location statue of PHPT1 using Laser scanning confocal microscope, and further studied the relationship of PHPT1 location with lamellipodia formation. Results： The pEGFP-N2-PHPT1 prokaryotic expression vectors was constructed successfully and the location of PHPT1 in lamellipodia was closely related to lamellipodia formation. Conclusion： C-terminal GFP fused PHPT1 were expressed in the nucleus and the cytoplasm, especially in the leading edge of lamellipodia, and this location was closely related to lamellipodia formation and cell mobility.

关 键 词：PHPT1 GFP融合表达 定位分析 层状伪足 

分 类 号：Q71[生物学—分子生物学]