Ca^(2+)依赖性磷脂酶A_2有效表达系统的构建

Construction of the Effective Expressing System for Calcium Dependent Phospholipase A_2 cDNA

为了构建Ca2 +依赖性磷脂酶A2 (CalciumdependentphospholipaseA2 ,cPLA2 )的有效表达系统 ,本实验应用SalⅠ消化cPLA2 -PMT2 重组体 ,电洗脱法回收cPLA2 片段并使其平末端化。应用NotⅠ消化PRC/CMV载体使其线性化 ,之后脱磷酸以防止自身环化。cPLA2 与线性化且脱磷酸载体PRC/CMV进行平末端连接 ,转染鼠巨噬细胞 ,扩增培养 ,收集细胞 ,提取RNA ,Northernblot检测cPLAmRNA。结果表明 :PRC/CMV可以做为cPLA2 cDNA的有效表达载体 ,cPLA2 -PRC/CMV表达系统的成功构建为研究各种因素对cPLA2

In order to construct an effective expression system for calcium dependent phospholipase A 2 (cPLA 2) cDNA,we got the cPLA 2 cDNA from PMT 2 vector which couldn't express cPLA 2 mRNA,and the cDNA fragment was recovered with electroelution method.We prepared the linearing and dephosphoring vector PRC/CMV by Not Ⅰ and CIP,ligated the cPLA 2 and PRC/CMV and transfected the recombinant into macrophage of mouse.Then the cells from the transfecting mouse were proliferated and RNA was isolated.The mRNA of cPLA 2 was measured with Northern blot.The results suggest that PRC/CMV is an effective vector for the expression of cPLAcDNA.