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

Construction of pcDNA3.1AM and expression of adrenomedullin in mammalian cells

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K56 2 细胞株上进行了体外表达实验。实验中采用RT PCR技术扩增AMcDNA片段 ,并将扩增的cDNA片段插入 pcDNA3 1真核表达质粒 ,构建成含AMcDNA的重组质粒 pcD NA3 1AM。用脂质体介导将该质粒转染培养的人白血病细胞K56 2 株。在转染的细胞中 ,用RT PCR检测证实有AMmRNA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 ,表明本实验中构建的重组pcDNA3 1AM载体能够在哺乳类细胞中表达AM。

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 pcDNA3 1, and in vitro expression of AM was carried out in cultured K 562 cell line. AM mRNA was amplified by RT PCR from the total RNA isolated from the adrenal glands of rats and was inserted into pcDNA3 1 vector to form pcDNA3 1AM, the recombinant pcDNA3 1AM was then transferred into cultured K 562 cell line by liposome. The expression of AM in pcDNA3 1AM transferred cell was identified by RT PCR and dot immunoblot assay. The results demonstrated that there were AM mRNA in the pcDNA3 1AM transferred K 562 cell line and AM peptides in the culturing medium, indicating that the recombinant pcDNA3 1AM vector can express AM in mammalian cell line.