| 人T细胞免疫球蛋白黏蛋白-4 cDNA全长真核表达载体的构建及其在16HBE细胞中的表达 |
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| 引用本文： | 吴小慧,李一荣,陈凤花,王琳,胡丽华. 人T细胞免疫球蛋白黏蛋白-4 cDNA全长真核表达载体的构建及其在16HBE细胞中的表达[J]. 免疫学杂志, 2010, 0(4) |
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| 作者姓名： | 吴小慧 李一荣 陈凤花 王琳 胡丽华 |
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| 作者单位： | 华中科技大学同济医学院附属协和医院检验科; |
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| 基金项目： | 国家自然科学基金资助项目(30672008) |
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| 摘 要： | 目的克隆人T细胞免疫球蛋白黏蛋白-4(TIM-4)基因cDNA,构建其真核表达载体pEGFP-C2-TIM-4,并转染16HBE细胞。方法根据GeneBank中人TIM-4 cDNA序列(编号:NM-138379.2),设计出带有EcoRI和BamHI的上下游引物,应用RT-PCR技术,从人骨髓中扩增出TIM-4基因编码区序列,克隆到pMD18-T载体,形成pMD18-T-TIM-4重组质粒,经菌落PCR,双酶切,测序鉴定获得人TIM-4基因cDNA片段,然后将其亚克隆入pEGFP-C2绿色荧光载体中,并通过菌落PCR,双酶切,测序鉴定其重组体。将测序正确的pEGFP-C2-TIM-4质粒转染人气道上皮细胞(16HBE),用实时定量PCR检测目的基因的表达。结果成功扩增出人TIM-4 cDNA全长1134 bp,经T-A克隆构建的pMD18-T-TIM-4重组质粒经菌落PCR,双酶切,测序证实载体中含有正确的TIM-4编码区片段。由此构建的真核表达载体pEGFP-C2-TIM-4,经菌落PCR扩增出1134 bp左右的片段,经双酶切后产生4.7 kb和1134 bp左右的2条带,DNA测序显示与GeneBan...
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| 关 键 词： | T细胞免疫球蛋白黏蛋白-4（TIM-4） 基因克隆 真核表达载体 聚合酶链反应（PCR） 转染 |
Construction of eukaryotic expression vector for human T cell immunoglobulin mucin protein-4 and its expression in 16HBE cells |
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| WU Xiaohui,LI Yirong,CHEN Fenghua,WANG Lin,HU Lihua. Construction of eukaryotic expression vector for human T cell immunoglobulin mucin protein-4 and its expression in 16HBE cells[J]. Immunological Journal, 2010, 0(4) |
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| Authors: | WU Xiaohui LI Yirong CHEN Fenghua WANG Lin HU Lihua |
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| Affiliation: | WU Xiaohui,LI Yirong,CHEN Fenghua,WANG Lin,HU Lihua Department of Clinical Laboratory,Union Hospital,Tongji Medical College,Huazhong University of Science , Technology,Wuhan 430022,China |
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| Abstract: | In this study,we construct a eukaryotic expression vector of the entirely coding human T cells immunoglobulin mucin protein -4(TIM-4) and transfect it into 16HBE cell line,for benefiting the further study on biological function of TIM-4.According to TIM-4 cDNA sequence in GeneBank(NO:NM-138379.2),we designed two primers which have sites of EcoRI and BamHI,respectively.The TIM-4 cDNA was amplified by RT-PCR from human bone marrow and then cloned into pMD18-T vector to obtain recombinant vector pMD18-T-TIM-4,... |
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| Keywords: | T cells immunoglobulin mucin protein-4 Gene cloning Eukaryotic expression vector Polymerase chain reaction Transfection |
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