| 血红素加氧酶-1融合蛋白PEP-1-HO-1的制备及在大鼠H9c2心肌细胞的转导 |
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| 引用本文： | 颜学滔,王焱林,王家宁,王成夭,何祥虎.血红素加氧酶-1融合蛋白PEP-1-HO-1的制备及在大鼠H9c2心肌细胞的转导[J].武汉大学学报(医学版),2010,31(4). |
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| 作者姓名： | 颜学滔 王焱林 王家宁 王成夭 何祥虎 |
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| 作者单位： | 1. 武汉大学中南医院麻醉科,湖北,武汉,430071
2. 郧阳医学院附属人民医院临床医学研究所,湖北,十堰,442000 |
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| 基金项目： | 国家自然科学基金资助项目 |
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| 摘 要： | 目的:制备血红素加氧酶-1(HO-1)融合蛋白PEP-1-HO-1,观察融合蛋白在大鼠H9c2心肌细胞的转导。方法:设计合成hHO-1 PCR引物,扩增hHO-1 cDNA片段;采用PCR靶向克隆法将扩增产物与含有相同酶切位点载体质粒pET15b-PEP-1进行重组,扩增质粒,双酶切鉴定和DNA测序;将测序和鉴定结果正确的重组质粒转化宿主菌Rosetta(DE3)pLysS,诱导表达融合蛋白PEP-1-HO-1,利用表达蛋白N端的组氨酸"标签"(His-tag)进行亲和层析纯化融合蛋白;SDS-PAGE和Western blotting对融合蛋白进行定性检测,Bradford法定量检测纯化后蛋白;融合蛋白孵育大鼠H9c2心肌细胞,免疫组化法观察融合蛋白在H9c2细胞的转导和分布,分光度法检测蛋白转导后的活性。结果:hHO-1 cDNA与pET15b-PEP-1重组成功,重组质粒pET15b-PEP-1-hHO-1经酶切鉴定含有hHO-1 cDNA,测序分析证实与GenBank提供的原始序列完全一致;重组质粒转化后经诱导和纯化得到了融合蛋白PEP-1-HO-1,浓度达到1.0 g/L;融合蛋白可以穿透H9c2心肌细胞胞膜,进入细胞后主要分布在胞质和胞核内并具有天然活性。结论:成功地构建了pET15b-PEP-1-hHO-1原核表达质粒,获得了可穿透H9c2心肌细胞膜的血红素加氧酶-1融合蛋白PEP-1-HO-1,为应用HO-1治疗缺血性疾病的研究奠定了基础。
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| 关 键 词： | 血红素加氧酶-1 PEP-1肽 融合蛋白 蛋白转导 H9c2细胞 |
Preparation of Fusion Protein PEP-1-HO-1 and Its Transduction into Rat H9c2 Cells |
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| YAN Xuetao,WANG Yanlin,WANG Jianing,WANG Chengyao,HE Xianghu.Preparation of Fusion Protein PEP-1-HO-1 and Its Transduction into Rat H9c2 Cells[J].Medical Journal of Wuhan University,2010,31(4). |
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| Authors: | YAN Xuetao WANG Yanlin WANG Jianing WANG Chengyao HE Xianghu |
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| Institution: | YAN Xuetao1,WANG Yanlin1,WANG Jianing2,WANG Chengyao1,HE Xianghu1 1Dept.of Anesthesiology,Zhongnan Hospital of Wuhan University,Wuhan 430071,China 2Institute of Clinical Medicine,People's Hospital of Yunyang Medical College,Shiyan 442000,China |
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| Abstract: | Objective: To prepare the HO-1 fusion protein PEP-1-HO-1 and transduct it into rat myocardial H9c2 cells.Methods: The primers of hHO-1 were designed and synthesized to amplify full-length hHO-1 cDNA by PCR cloning.After double-enzyme digestion,the linearized pET15b-PEP-1 and hHO1 cDNA was ligated with PCR cloning reaction to construct pET15b-PEP-1-hHO-1.The recombinant plasmid was verified by PCR and DNA sequencing,and transformed into Rosetta(DE3)pLysS host bacteria which was induced to obtain fusion prote... |
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| Keywords: | Heme Oxygenase-1 PEP-1 Fusion protein Protein transduction H9c2 Cardiomyocytes |
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