胞内型磷脂酶A2基因亚克隆与基因表达

Subcloning and Gene Expression of Cytosolic Phospholipace

目的 研究花生四稀酸在细胞中的释放及前列腺素的合成，构建与花生四稀酸释放相关的表达系统。即胞内型磷脂酶Ａ２（ｃｙｔｏｓｏｌｉｃ ｐｈｏｓｐｈｏｌｉｐａｓｅ ｃＰＬＡ２）ｃＤＮＡ－ｐＲＣ／ＣＭＶ。方法 从克隆载体ｐＭＴ２用Ｓａｌ１制备ｃＰＡＬ２ｃＤＮＡ；平末端连接ｃＰＡＬ２－ｐＲＣ／ＣＭＶ；转染鼠巨噬细胞及筛选正向阳性克隆；ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测ｃＰＬＡ２ ｃＤＮＡ基因表达。结果

Objective To study the release of arachidonic acid and the biosynthesis of prostaglandins,the expression system of pRC/CMV inserting cytosolic phospholipase cDNA was constituted.Methods to prepare cPLA 2 cDNA from pMT 2 plasmid with Sal1;bluut end ligation of cPLA 2 cDNA-pRC/CMV;transfect the recombinant into maerophage-like cell and choose the sence subcloning;checc the expression of cPLA 2 cDNA by Northeen blot.Results The expression was showed on the autoradiography film.pRC/CMV is an effective vector for hcPLA 2 cDNA expression this work lays a foundation for further study on the expression of cPLA 2 and fransformation from unsaturation fatty acid to prostaglandins.