【摘要】 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更多还原

【Abstract】 AIM: To construct a eukaryotic expression vector for cytotoxic T lymphoc yte antigen-4 (CTLA4) gene fused with enhanced green fluorescent pro tein (EGFP) gene and to analyze the expression and subcellular localization of t he fusion protein in K562 cells. METHODS: The human CTLA4 gene w as cloned by RT-PCR and was then inserted into plasmid pEGFP-N1 to construct the expression vector for CTLA4-EGFP fusion gene. The expression and subcellula r localization of the fusion protein in transfected K562 cells were analyzed by flow cytometry and confocal microscopy, respectively. RESULTS: The cDNA of CTLA4 gene was cloned from human peripheral blood cells. The express ion vector for CTLA4-EGFP fusion gene was constructed by PCR through introducin g a Kozak sequence before the initiation site and deleting the stop codon. Flow cytometry analysis revealed that the proportion of both CTLA4 + and EGFP + K56 2 cells was 16% at 24 h after the transfection. Confocal microscopy observation showed that the expressed fusion protein was mainly distributed in the intracell ular compartments and had small part on the cell membrane. In contrast, only EGF P expression could be detected in K562 cells transfected with empty vector with diffuse distribution in the cells. Moreover, the expression level of the fusion protein on the cells increased to 29% after stimulation with phorbol ester and ionomycin. In addition, the fusion protein inside the cells tended to move towa rds and fused with the cell membrane. CONCLUSION: The expression vector for CTLA4-EGFP fusion gene has been constructed successfully and expres sed in K562 cells. The expressed fusion protein has similar subcellular localiza tion and transport characteristics to native CTLA4 in activated T cells.更多还原