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为了构建分泌型磷脂酶A2(SECRETARY PHOSPHOLIPASE A2,SPLA2) CDNA的有效表达系统,该文从无效表达的重组体SPLA2 － PGEM7 中制备SPLA2 片段,将其插入中间载体BLUESCRIPTBSSKR的ECOR1 位点,利用SPLA2－ BSSK重组体转化DH1 感受态细胞,筛选ANTISENSESPLA2－ BSSK 重组体,应用XBAL、HIND3 双酶消化该重组体并回收带有不同粘性末端的SPLA2 片段,因XBAL、HIND3 消化载体PRC/CMV,使之线性化并带有XBAL、HIND3 的粘性末端.SPLA2 定向插入PRC/CMV 载体,构建成具有CMV 强启动子的表达系统.重组体转染鼠巨噬细胞,扩增培养,从细胞中提取总RNA,NORTHERN 杂交法检测SPLA2 MRNA.结果表明PRC/CMV 可以用于SPLA2 表达及各种因素影响表达的研究.“,”To construct an effective expression system for secretory phospholipase A2 (sPLA2) cDNA,we got the sPLA2 cDNA from pGEM7 vector which couldn't express sPLA2 mRNA,and then inserted sPLA2 fragment into the second vector Bluescript BSSK in EcoRl site.We transfered sPLA2-BSSK recombinant into DH1,screened antisense sPLA2-BSSK,digested it by Xbal,HindⅢ and recovered sPLA2 fragment with different cohesive ends.We also cut the vector pRc/CMV with two enzymes to make pRc/CMV be lineared.After cloning sPLA2 into pRc/CMV,the sPLA2-pRc/CMV recombinant was transfected into macrophage of mouse.The cells of mouse were multiplied and RNA was isolated,mRNA of sPLA2 was checked with Northern Blot.The results showed that pRc/CMV was an effective vector for expression study of sPLA2.