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目的:制备分泌型的磷脂酶A2 G13(group XIII se-cretedphos pholipaseA2,PLA2G13)的多克隆抗体(pAb)和单克隆抗体(mAb),并对抗体进行特性鉴定。方法:以人正常肝cDNA文库为模板,构建重组表达质粒pGEX-4T-1-PLA2G13(即PLA2G13-GST)和pET-32a-PLA2G13(即PLA2G13-His)。PLA2G13-GST融合蛋白作为免疫原制备兔pAb和鼠mAb。采用Western blot鉴定兔抗人PLA2G13 pAb的特异性。采用Western blot和Immunohistochemistry鉴定鼠抗人PLA2G13 mAb的特异性。结果:PLA2G13-GST和PLA2G13-His融合蛋白在均大肠杆菌中以包涵体形式大量表达。Western blot鉴定表明,兔抗人PLA2G13 pAb可特异地识别HepG2细胞系中相对分子质量(Mr)约19700的蛋白,与文献报道中PLA2G13的实际Mr相符。通过筛选共获得8株可稳定分泌抗PLA2G13的杂交瘤细胞株,Western blot鉴定表明6株可识别重组人PLA2G13蛋白,免疫组织化学鉴定显示2株可特异性识别正常肝组织中的PLA2G13蛋白。结论:成功地制备了抗人PLA2G13兔pAb和鼠mAb,为进一步研究PLA2G13的功能提供了特异性检测工具。
OBJECTIVE: To prepare polyclonal antibody (pAb) and monoclonal antibody (mAb) of secreted phospholipase A2 G13 (group XIII se-cretedphos pholipase A2, PLA2G13), and to characterize the antibody. Methods: Recombinant plasmids pGEX-4T-1-PLA2G13 (PLA2G13-GST) and pET-32a-PLA2G13 (PLA2G13-His) were constructed by using human normal liver cDNA library as a template. Rabbit pAb and murine mAbs were prepared using PLA2G13-GST fusion protein as an immunogen. The specificity of rabbit anti-human PLA2G13 pAb was identified by Western blot. The specificity of the murine anti-human PLA2G13 mAb was identified by Western blot and Immunohistochemistry. Results: PLA2G13-GST and PLA2G13-His fusion protein were highly expressed as inclusion bodies in Escherichia coli. Western blot showed that rabbit anti-human PLA2G13 pAb could specifically recognize about 19,700 proteins with relative molecular mass (Mr) in HepG2 cell line, which accorded with the actual Mr2 in PLA2G13 reported in the literature. A total of 8 hybridoma cell lines stably secreting anti-PLA2G13 were obtained by screening. Western blot showed that 6 strains could recognize recombinant PLA2G13 protein. Immunohistochemistry showed that 2 strains could specifically recognize PLA2G13 protein in normal liver tissue. Conclusion: The anti-human PLA2G13 rabbit pAb and murine mAb were successfully prepared, which provided a specific detection tool for further study on the function of PLA2G13.