| 定向克隆构建RAB5A基因真核细胞表达载体 |
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| 引用本文： | 陈宇,张泽坤,邹嵘. 定向克隆构建RAB5A基因真核细胞表达载体[J]. 哈尔滨医科大学学报, 2000, 34(2): 79-81 |
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| 作者姓名： | 陈宇 张泽坤 邹嵘 |
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| 作者单位： | |
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| 基金项目： | 国家自然科学基金!( 3 9970 3 96)，黑龙江省自然科学基金! (D980 9) |
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| 摘 要： | 目的 探讨ＲＡＢ５Ａ作为蛋白质人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（－）０ＲＡＢ５Ａ。结果 测定分析证实了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２３ＫＤ的蛋白质
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| 关 键 词： | RAB5A基因 定向克隆 蛋白电泳 肿瘤转移 肺肿瘤 |
Construction of RAB5A gene eukaryotic expression vector by direct cloning |
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| CHEN Yu,,ZOU Rong,et alZHANG Ze-kun. Construction of RAB5A gene eukaryotic expression vector by direct cloning[J]. Journal of Harbin Medical University, 2000, 34(2): 79-81 |
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| Authors: | CHEN Yu ZOU Rong et alZHANG Ze-kun |
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| Affiliation: | CHEN Yu,,ZOU Rong,et al;(1.Medical Genetics Laboratory,Harbin Medical University);ZHANG Ze-kun;(The Se cond Affiliated Hospital,Harbin Medical University,Harbin 150086,China) |
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| Abstract: | 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. |
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| Keywords: | human RAB5A gene direct cloning protein electrophoresis immunohistochemistry |
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