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目的 :构建增强型绿色荧光蛋白 (EGFP)与CTLA4融合蛋白真核表达载体 ,分析其在K562细胞中的表达和亚细胞定位。方法 :以RT PCR方法克隆人CTLA4基因 ,构建CTLA4 EGFP融合蛋白的表达载体。以其转染K562细胞后 ,以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果 :从人外周血细胞中克隆到CTLA4基因的cDNA。通过PCR方法在起始位点前加入Kozak序列并删除终止密码 ,成功地构建CTLA4 EGFP融合蛋白表达载体。以该质粒转染K562细胞 2 4h后 ,CTLA4和EGFP双阳性细胞的百分率为 16%。表达的融合蛋白主要分布于胞内 ,细胞膜上分布较少。相反 ,转染空载体的细胞仅表达EGFP ,且其在细胞内呈弥散样分布。以佛波醇酯加离子霉素刺激后 ,CTLA4 EGFP融合蛋白在细胞表面表达水平升高到 2 9% ,且分布于胞内的融合蛋白向细胞膜靠近并与质膜融合 ;而转染空载体的细胞内EGFP的分布无明显改变。结论 :成功地构建CTLA4 EGFP融合蛋白表达载体 ,并在K562细胞中得到表达。表达的融合蛋白与天然CTLA4在活化T细胞中的定位和转运特点相似
OBJECTIVE: To construct eukaryotic expression vector of enhanced green fluorescent protein (EGFP) and CTLA4 fusion protein and analyze its expression and subcellular localization in K562 cells. Methods: The human CTLA4 gene was cloned by RT PCR and the CTLA4 EGFP fusion protein was constructed. After transfected into K562 cells, the expression of fusion protein and its subcellular localization were analyzed by flow cytometry and laser confocal microscopy. Results: cDNA of CTLA4 gene was cloned from human peripheral blood cells. The Kozak sequence was added before the start site by PCR and the stop codon was deleted to construct the CTLA4 EGFP fusion protein expression vector successfully. After transfecting K562 cells with this plasmid for 24 h, the percentage of double positive CTLA4 and EGFP cells was 16%. The expressed fusion proteins mainly distributed in the intracellular, with less distribution on the cell membrane. In contrast, cells transfected with empty vector only expressed EGFP, and they were diffusely distributed within the cells. After stimulation with phorbol ester and ionomycin, the expression level of CTLA4 EGFP fusion protein increased to 29% on the cell surface, and the intracellular fusion protein approached the cell membrane and fused with the plasma membrane. However, The intracellular EGFP distribution did not change significantly. Conclusion: CTLA4 EGFP fusion protein expression vector was successfully constructed and expressed in K562 cells. The expressed fusion protein has similar characteristics to the localization and trafficking of native CTLA4 in activated T cells