| GST-hPBD融合蛋白的克隆、表达及活化Rac1蛋白的分析 |
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| 引用本文： | 韩雅玲,孟子敏,康建,刘海伟,闫承慧,王士雯. GST-hPBD融合蛋白的克隆、表达及活化Rac1蛋白的分析[J]. 医学争鸣, 2004, 25(13): 1218-1221 |
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| 作者姓名： | 韩雅玲 孟子敏 康建 刘海伟 闫承慧 王士雯 |
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| 作者单位： | 沈阳军区总医院全军心血管病研究所心内科,辽宁,沈阳,110016;解放军总医院老年心血管研究所,北京,100850 |
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| 基金项目： | 国家重点基础研究发展计划(973计划) |
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| 摘 要： | 目的:制备GST-hPBD融合蛋白,探讨活化的Rac1在血管内皮细胞中的表达. 方法:采用RT-PCR方法克隆与活化Rac1相结合的hPBD基因片段,构建pGEX-2T/hPBD原核表达载体,并纯化GST-hPBD融合蛋白;应用pull down测定血管内皮细胞中活化Rac1的蛋白表达. 结果:基因测序证实原核表达载体pGEX-2T/hPBD序列正确,并在大肠杆菌中高效表达,纯化得到纯度约75% GST-hPBD融合蛋白,其Mr为36 000;进一步分析证实血管内皮细胞可表达活化的Rac1蛋白. 结论:成功构建了人pGEX-2T/hPBD原核表达载体,纯化了GST-hPBD融合蛋白,并检测到血管内皮细胞中活化Rac1蛋白的表达.
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| 关 键 词： | 结构域 基因 融合蛋白 纯化 |
| 文章编号： | 1000-2790(2004)13-1218-04 |
| 修稿时间： | 2004-02-16 |
Cloning and expression of GST-hPBD fusion protein for activated Rac1 protein analysis |
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| HAN Ya Lin ,MENG Zi Min ,KANG Jian ,LIU Hai Wei ,YAN Cheng Hui ,WANG Shi Wen. Cloning and expression of GST-hPBD fusion protein for activated Rac1 protein analysis[J]. Negative, 2004, 25(13): 1218-1221 |
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| Authors: | HAN Ya Lin MENG Zi Min KANG Jian LIU Hai Wei YAN Cheng Hui WANG Shi Wen |
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| Affiliation: | HAN Ya Lin 1,MENG Zi Min 1,KANG Jian 1,LIU Hai Wei 1,YAN Cheng Hui 1,WANG Shi Wen 2 1Department of Cardiovasology,General Hospital of Chinese PLA,Shenyang Command,Shenyang 110016,China,2Institute of Cardiovascular Diseases,General Hospital of Chinese PLA,Beijing 100850,China |
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| Abstract: | AIM: To induce and express the GST hPBD fusion protein and to study the expression of Rac1 in endothelial cell line ECV304. METHODS: The gene fragment of hPBD was amplified by RT PCR and cloned into the pGEX 2T prokaryotic expression vector, and the GST hPBD fusion protein was purified. The activated Rac1 protein was detected with GST hPBD by pull down assay. RESULTS: DNA sequencing confirmed that the expression vector of pGEX 2T/hPBD was constructed successfully and fusion protein was expressed effectively in Escherichia coli , with about 75% of the GST hPBD fusion protein ( M r 36 000 ). Pull down assay found the activated Rac1 protein in ECV304 cells. CONCLUSION: The pGEX 2T prokaryotic expression vector is successfully constructed and the GST hPBD fusion protein is purified. Activated Rac1 protein is detected in vascular endothelial cell ECV304. This study lays the basis for the research on the relationship between the Rac1 activity and vascular endothelial cell permeability. |
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| Keywords: | domain gene fusion protein purification |
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