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目的构建人脂蛋白相关磷脂酶A2(Lp-PLA2)的表达载体,表达Lp-PLA2重组蛋白,并制备其单克隆抗体(mAb).方法合成Lp-PLA2基因并插入融合表达载体pBV220的多克隆位点,构建重组表达质粒pBV220/Lp-PLA2.以重组质粒转化大肠杆菌DH5α,筛选阳性重组子诱导目的蛋白的表达,表达产物的免疫学活性用Western blot进行鉴定.以基因重组的Lp-PLA2蛋白为抗原,常规方法免疫BALB/c小鼠,取其脾细胞与NS-1细胞融合,获得稳定分泌Lp-PLA2 mAb的杂交瘤细胞株,ELISA检测mAb的效价;Western blot检测mAb的特异性.结果酶切鉴定和DNA测序显示Lp-PLA2重组表达载体中含有人Lp-PLA2全长编码序列.将该重组载体转化入大肠杆菌DH5α中表达所得蛋白经Western blot验证为目的蛋白,筛选出2株能稳定分泌特异性抗Lp-PLA2的mAb杂交瘤细胞株.结论该研究成功地构建了Lp-PLA2表达载体、表达出Lp-PLA2重组蛋白,制备出抗Lp-PLA2 mAb,为进一步用于Lp-PLA2的体外免疫学检测奠定了基础.“,”Objective To prepare monoclonal antibody(mAb) against recombinant human lipoprotein-associated phospholipase A2(Lp-PLA2).Methods The full length gene encoding Lp-PLA2 was synthesized and inserted into expression plasmid pBV220 to construct recombinant plasmid pBV220/Lp-PLA2.The recombinant plasmid was transformed into E.coli DH5α which then expressed Lp-PLA2.The immunological activity of the expressed Lp-PLA2 was analyzed by Western blot.Recombinant human Lp-PLA2 protein was used as antigen to immunize BALB/c mice.Monoclonal anti-bodies against Lp-PLA2 were prepared by normal hybridoma technology.The antibody titer of mAbs was determined by ELISA.Specificity of mAbs was analyzed by Western blot.Results Human Lp-PLA2 gene was synthesized and confirmed by DNA sequencing.Positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing.Westem blot analysis showed that the Lp-PLA2 protein could be recognized by an anti-Lp-PLA2 antibody.Two hybridmas producing antibodies against Lp-PLA2 were obtained.Conclusion The recombinant expression plasmid of Lp-PLA2 was constructed successfully and expressed in E.coli.The method of ELISA established to test serum Lp-PLA2 is useful.The Lp-PLA2 mAb using Lp-PLA2 as antigen prepared in this paper can be used for immunoassay in vitro.