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目的 探讨RAB5A作为蛋白质入胞信号传导调控者在肿瘤转移中的作用及影响。方法 采用双酶切、定向克隆技术建立了人RAB5A基因真核细胞表达载体pcDNA3.1(- ) -RAB5A。结果 测序分析证实了RAB5A基因正向插入pcDNA3.1(- )表达载体 ;免疫组化分析表明转染了pcDNA3.1(- ) -RAB5A表达载体的AGZY83 a细胞系细胞内的荧光亮度明显强于转染前 ,蛋白电泳显示近 2 3KD的蛋白质含量增高 ,表明重组质粒在细胞内工作良好。结论 双酶切、定向克隆技术是一种建立人RAB5A基因真核细胞表达载体的有效方法
Objective To investigate the role and influence of RAB5A as a regulator of protein signal transduction in tumor metastasis. METHODS: The human RAB5A gene eukaryotic cell expression vector pcDNA3.1(-)-RAB5A was established by double enzyme digestion and directional cloning. Results Sequencing analysis confirmed that the RAB5A gene was positively inserted into the pcDNA3.1 (-) expression vector; immunohistochemical analysis showed that the fluorescence intensity of the cells transfected with the pcDNA3.1 (-)-RAB5A expression vector was significantly stronger than that of the AGZY83 a cell line. Prior to transfection, protein electrophoresis showed an increase in the protein content of the near 23KD, indicating that the recombinant plasmid worked well in cells. Conclusion The double enzyme digestion and directional cloning technique is an effective method to establish the eukaryotic expression vector of human RAB5A gene.