| 反义PCDNA3.1(+)/TIMP-1真核表达载体的构建与鉴定 |
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| 引用本文： | 李睿,郑勇,周婷,奚海林,潘泽民,杨军.反义PCDNA3.1(+)/TIMP-1真核表达载体的构建与鉴定[J].天津医药,2007,35(5):366-368. |
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| 作者姓名： | 李睿 郑勇 周婷 奚海林 潘泽民 杨军 |
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| 作者单位： | 832008,石河子大学医学院第一附属医院消化科 |
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| 基金项目： | 新疆生产建设兵团科技攻关项目 |
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| 摘 要： | 目的：构建反义TIMP-1真核细胞表达质粒。方法：用Trizol Reagent抽提肝组织中总RNA．采用两对套式引物，以逆转录巢式PCR方法扩增TIMP-1目的片段，双酶切纯化PCR产物及PCDNA3.1（＋）真核细胞表达质粒。再将TIMP-1反向插入PCDNA3.1（＋）线性质粒，即构建成反义TIMP-1／PCDNA3.1（＋）真核细胞表达质粒，经酶切、PCR及DNA测序鉴定。结果：DNA测序结果与预期目的片段序列一致。结论：反义TIMP-1／PCDNA3．1（＋）真核细胞表达质粒构建成功。
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| 关 键 词： | 真核细胞质 遗传载体 基因表达 质粒 寡核苷酸类 反义 逆转录聚合酶链反应 |
| 修稿时间： | 2006-05-092006-10-23 |
Construction and Identification of Antisense TIMP-1 Eukaryotic Expressing Plasmids |
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| LI Rui,ZHENG Yong,ZHOU Ting,XI Hailin,PAN Zemin,YANG Jun.Construction and Identification of Antisense TIMP-1 Eukaryotic Expressing Plasmids[J].Tianjin Medical Journal,2007,35(5):366-368. |
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| Authors: | LI Rui ZHENG Yong ZHOU Ting XI Hailin PAN Zemin YANG Jun |
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| Institution: | Department of Digest, First Affiliated Hospital, Shihezi University Medical College, Xinjiang 832008, China |
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| Abstract: | Objective: To construct antisense TIMP-1 eukaryotic expressing plasmids and transfect in vivo. Methods:Trizol reagent was used to extract total RNA in rat liver. The proper nested primers of TIMP-1 were chosen and synthesized. RT-nest-PCR and gene recombinant techniques were used to construct the fragment of TIMP-1. After purification, the PCR products of TIMP-1 and eukaryotic expressing plasmid PCDNA3.1(+) were cut, TIMP-1 were inserted reversely into PCDNA3.1(+), and then transferred into JM-109 strain. Enzyme-cutting and PCR identification and DNA autosequencing were used to prove the successful construction of antisense TIMP-1 eukaryotic expressing plasmid. Results: Sequence analysis showed the same sequence as expected. Conclusion: Construction of antisense TIMP-1 eukaryotic expressing plasmids is successful. |
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