Sema4C及其相互作用蛋白GIPC的亚细胞定位及荧光共定位研究

目的：观察脑信号蛋白Sema4C及其相互作用蛋白GIPC的亚细胞定位及两者的荧光共定位情况，为明确Sema4C和GIPC在亚细胞水平的相互作用提供佐证。方法：将Sema4C的基因编码区全长、胞外段和胞内段分别构建到pEGFPNl和pEGFPCI表达载体中，将GIPC编码区基因构建到pDsRed-C1表达载体中，分别转染HEK293细胞，观察亚细胞定位；将pEGFPNl-Sema4C和pDsRed-GIPC分别共转染HEl（293和COS7细胞，观察两者的荧光共定位情况。结果：酶切鉴定及测序结果表明重组载体构建正确，Sema4C蛋白全长和胞外段呈跨膜分布，而胞内段在全细胞中呈弥散样分布；GIPC在胞浆内呈斑块状聚集分布；pEGFPNl-Sema4C和pDsRed-GIPC存在荧光共定位区域。结论：Sema4C主要在胞膜和胞浆内表达，GIPC主要在胞浆内呈斑块样聚集分布；Sema4C和GIPC之间存在荧光共定位。

Studies on Subcellular Localization and Fluorescent Colocalization of Sema4C and its Interacting Protein GWC

Objective： To investigate the subcellular localization and fluorescent colocalization of Sema4C and its interacting protein GIPC, so as to provide evidences of protein-protein interaction between Sema4C and GIPC at subcellular level. Methods： The full length（FL）, extracellular domain（ED） and intercellular domain（ID） of Sema4C encoding sequences were subcloned and constructed into the expressive vectors of pEGFPN1 and pEGFPC1 respectively. GIPC encoding sequence was subcloned and constructed into the expressive vector of pDsRed-C1. HEK293 cells were transfected with the recombinant vectors and control vectors to observe the subcellular localization of Sema4C and GIPC. Besides, pEGFPN1- Sema4C and pDsRed-GIPC vectors were both transfected into HEK293 and COS7 cells respectively to investigate the fluo- rescent colocalization of Sema4C and GIPC by confocal microscope observation. Results： The recombinant expression plasmids were confirmed by restriction enzyme digestion. Data showed that Sema4C-FL and Sema4C-ED fused with EGFP were distributed with a transmembrane pattern while Sema4C-ID was distributed uniformly in whole cells in transfected HEK293 cells. The red fluorescence of GIPC fused protein could be seen in the cytoplasm of transfected HEK293 cells with a plaque-like pattern. It was shown that the expression of pEGFPN1-Sema4C and pDsRed-GIPC existed fluorescent colocalization areas in cotransfected cells. Conclusion： The expression of Sema4C was mainly distributed in the cell mem- brane and cytoplasm in the transfected cells. The expression of GIPC was mainly distributed in the cytoplasm with a plaque-like pattern. It was existed fluorescent colocalization areas between Sema4C and GIPC in transfected cells.