定向克隆构建RAB5A基因真核细胞表达载体  

Construction of RAB5A gene eukaryotic expression vector by direct cloning

作　　者：陈宇[1] 张泽坤[2] 邹嵘[1] 冯会臣[1] 王柏秋[1] 阎承慧[1] 李钰[1] 李璞[1] 

机构地区：[1]哈尔滨医科大学医学遗传学研究室 [2]哈尔滨医科大学附属第二医院,黑龙江哈尔滨150086

出　　处：《哈尔滨医科大学学报》2000年第2期79-81,共3页Journal of Harbin Medical University

基　　金：国家自然科学基金!( 3 9970 3 96) ;黑龙江省自然科学基金! (D980 9)

摘　　要：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。Objective To construct a recombinant eukaryotic plasmid pcDNA3.1(-)-RAB5A for the coming research on the molecular mechanism of RAB5A gene in tumor metastasis through transfection. Methods Double enzyme digestion and direct cloning were employed to construct the recombinant eukaryotic plasmid pcDNA3.1(-)-RAB5A.Results The structure of the recombinant plasmid were confirmed by cycle sequencing.Compared with the former cell line,protein electrophoresis showed that a highly expressed protein in size of about 23KD(coded by RAB5A gene) was synthesized in transfected AGZY83 a cell line,and more FITC fluorescence were detected by RAB5A specific antibody with the method of immunohistochemistric stain,suggesting that the recombinant eukaryotic plasmid pcDNA3.1(-)-RAB5A works well in the two cell lines.Conclusion Direct cloning is a effective method of recombinant eukaryotic plasmid construction.

关 键 词：RAB5A基因 定向克隆 蛋白电泳 肿瘤转移 肺肿瘤 

分 类 号：R734.202[医药卫生—肿瘤] R73－37[医药卫生—临床医学]