| 慢性粒细胞白血病bcr/abl的糖基化磷脂酰肌醇锚定修饰及在COS-7细胞膜上的表达 |
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| 引用本文： | 陶崑,王东,曾建明,黄世峰,陈新敏,黄宗干,冯文莉.慢性粒细胞白血病bcr/abl的糖基化磷脂酰肌醇锚定修饰及在COS-7细胞膜上的表达[J].第三军医大学学报,2008,30(8):717-720. |
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| 作者姓名： | 陶崑 王东 曾建明 黄世峰 陈新敏 黄宗干 冯文莉 |
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| 作者单位： | 重庆医科大学基础医学院免疫学教研室,重庆,400016;重庆医科大学临床血液学教研室,临床检验诊断学省部共建教育部重点实验室,重庆,400016;重庆医科大学附属第一医院血液科,重庆,400016 |
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| 摘 要： | 目的 构建与糖基化磷脂酰肌醇(glycosyl phosphatidyl inositol,GPI)锚定序列相连的bcr/abl真核表达质粒,并检测其在COS-7细胞中基因和膜蛋白的表达.方法 以编码慢性粒细胞白血病bcr/abl融合蛋白全长序列的migP210质粒为模板,通过PCR扩增获得654 bp的bcr/abl融合基因片段,双酶切后定向插入真核表达载体pBudCF4.1中,经鉴定获得重组质粒pBB.同时用RT-PCR扩增人外周血淋巴细胞CD24分子中的GPI获得约243 bp的锚定序列,双酶切后克隆人pBB质粒中与ber/abl基因下游羧基端相连,获得重组质粒pBBG.在多聚阳离子介导下将pBBG质粒转染COS-7细胞,采用RT-PCR和Western blot检测bcr/abl融合基因和蛋白的表达.结果 经酶切和测序鉴定证实,由GPI锚定连接的ber/abl融合基因插入片段正确;RT-PCR检测到转染细胞中有bcr/abl融合基因,Western blot检测到转染细胞膜上有hcr/abl融合蛋白的表达.结论 成功构建由GPI锚定修饰的bcr/abl重组质粒pBBG,并能在COS-7细胞膜上表达出bcr/abl融合蛋白.
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| 关 键 词： | bcr/abl融合基因 GPI 真核表达 慢性粒细胞白血病 |
| 文章编号： | 1000-5404(2008)08-0717-04 |
| 修稿时间： | 2007年8月15日 |
Construction of GPI-anchored bcr/abl and its expression on COS-7 cells membrane |
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| TAO Kun,WANG Dong,ZENG Jian-ming,HUANG Shi-feng,CHEN Xin-min,HUANG Zong-gan,FENG Wen-li.Construction of GPI-anchored bcr/abl and its expression on COS-7 cells membrane[J].Acta Academiae Medicinae Militaris Tertiae,2008,30(8):717-720. |
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| Authors: | TAO Kun WANG Dong ZENG Jian-ming HUANG Shi-feng CHEN Xin-min HUANG Zong-gan FENG Wen-li |
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| Affiliation: | TAO Kun1,WANG Dong2,ZENG Jian-ming2,HUANG Shi-feng2,CHEN Xin-min2,HUANG Zong-gan3,FENG Wen-li2(1Department of Immunology,Faculty of Basic Medical Sciences,2 Department of Clinical Hematology,Key Laboratory of Laboratory Medical Diagnosis of Ministry of Education,Faculty of Laboratory Medicine,3Department of Hematology,The First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China) |
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| Abstract: | Objective To construct a recombinant eukaryotic expression plasmid of glycosylphosphatidyl inositol(GPI)-anchored bcr/abl and explore its expression at mRNA and protein level.Methods The gene fragment encoding bcr/abl was amplified by PCR using the plasmid containing the cDNA sequence of P210 as template and then inserted into a eukaryotic expression vector pBudCE4.1.The constructed recombinant plasmid pBudCE4.1-bcr/abl was identified by restriction analysis and DNA sequencing.Lymphocytes were isolated from... |
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| Keywords: | GPI |
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