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目的：选择适当的载体，启动Ca~(2+)依赖分泌型磷脂酶A2（SecretaryphospholipaseA2sPLA2）cDNA的表达，为进一步研究各种因素对该酶活性的影响奠定基础。方法：首先从sPLA2-pGEM7重组体（这一重组体不能在转染的真核细胞中表达）获得人sPLA2cDNA，克隆该片断进入中间载体BSSK（使aPLA2cDNA两端出现XbaI、Hind3酶切位点），选择antisensesPLA3-BSSK重组体进行扩增，双酶消化上述重组体及PRC／CMV，电洗脱回收aPLA2片断及线性化的载体PRC／CMV，定向连接sPLA2-PRC/CMV，重组体转染鼠巨噬细胞，筛选，扩增阳性克隆；提取RNA，Northem杂交法检测sPLA22mRNA。结果：应用定向克隆方法，成功地制备了sPLA2-PRC/CMV重组体。用这一重组质粒转染鼠巨噬细胞后，Northem杂交检测到了sPLA2－mRNA的表达。结论：SPLA_2－PRC／CMV是一有效的表达系统。
OBJECTIVE: To select the appropriate vector to initiate the expression of Ca 2+ -secretory phospholipase A2 (cDNA of Secretary phospholipase A2PLA2), and to lay a foundation for further study on the effect of various factors on the activity of this enzyme. METHODS: Human sPLA2 cDNA was first obtained from sPLA2-pGEM7 recombinant (this recombinant was not expressed in transfected eukaryotic cells) and cloned into the intermediate vector BSSK (XbaI, Hind3 restriction sites flanked by aPLA2 cDNA) , Selected antisensesPLA3-BSSK recombinant amplification, double enzyme digestion of these recombinants and PRC / CMV, electroporation recovery aPLA2 fragments and linearized vector PRC / CMV, directional connection sPLA2-PRC / CMV, the recombinant transfected mice Macrophages, screening, amplification of positive clones; RNA extraction, Northem hybridization assay sPLA22 mRNA. Results: The sPLA2-PRC / CMV recombinant was successfully prepared by directional cloning method. After transfecting murine macrophages with this recombinant plasmid, sPLA2-mRNA expression was detected by Northem hybridization. Conclusion: SPLA_2-PRC / CMV is an effective expression system.