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重组肾上腺髓质素表达载体的构建及在哺乳动物细胞中的表达

王小芳, 邵颖, 田德志, 姚泰, 陆利民

复旦大学上海医学院生理与病理生理学系，上海 200032

摘要

为探索通过体内表达肾上腺髓质素(adrenomedullin, AM)治疗高血压和慢性心衰的可能性，本实验构建了重组AM真核表达载体，并在无内源性AM表达的凡62细胞株上进行了体外表达实验。实验中采用RT-PCR技术扩增AM cDNA片段，并将扩增的cDNA片段插入pcDNA3. 1真核表达质粒，构建成含AM cDNA的重组质粒pc DNA3. 1 AM。用脂质体介导将该质粒转染培养的人白血病细胞蛛62株。在转染的细胞中，用RT-PCR检测证实有AMm RNA存在;用斑点免疫分析方法检测转染细胞的培养液上清，证实有AM多肽存在，表明实验中构建的重组
pcDNA3. 1 AM载体能够在哺乳类细胞中表达AM.

关键词： 肾上腺髓质素; K_(562)细胞; 基因表达; 脂质体

Construction of pcDNA3.lAM and expression of adrenomedullin in mammalian cells

WANG Xiao- Fang, SHAO Ying, Tian Dezhi, Yao Tai, LU Li- Min

Department of Physiology and Pathaphysiology，Shanghai Medical College，Fudan University，Shanghai 200032

Abstract

The newly discovered endogenous vasodilating and diuretic peptide adrenomedullin(AM)was considered to be of attractive value in clinical treatment of hypertension and congestive heart failure.In order to explore the treatment of cardiovascular diseases by expressing AM in vivo，AM cDNA was
inserted into mammalian expressing vector pcDNA31，and in vitro expression of AM was carried out in cultured K562 cell line AM mRNA was amplified by RT-PCR from the total RNA isolated from the adrenal glands of rats and was inserted into pcDNA31 vector to form pcDNA3I AM，the recombinant
pc DNA31 AM was then transferred into cultured K562 cell line by posome .The expression of AM in pc DNA3.1 AM transferred cell was identified by RT- PCR and dot i m munoblot assay.The results demonstrated that there were AM mRNA in the pcDNA31 ANPtransferred K562 cell line and AM peptides in the culturing medium，indicating that the recombinant pcDNA3 1 AM vector can express AM in mammalian cell line.

Key words: adreno medullin;;K_(562) cell line;

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引用本文：

王小芳, 邵颖, 田德志, 姚泰, 陆利民. 重组肾上腺髓质素表达载体的构建及在哺乳动物细胞中的表达[J]. 生理学报 2003; 55 (1): .

WANG Xiao- Fang, SHAO Ying, Tian Dezhi, Yao Tai, LU Li- Min. Construction of pcDNA3.lAM and expression of adrenomedullin in mammalian cells. Acta Physiol Sin 2003; 55 (1): (in Chinese with English abstract).