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目的:制备PON2(paraxonase2)单克隆抗体(mAb),并进行初步鉴定。方法:利用生物信息学方法分析人类PON2蛋白序列,选取与小鼠同源性低,而免疫原性与亲水性均较强的片段,构建重组表达质粒pGEX-4T-1-PON2和PET-32a-PON2,GST-PON2和HIS-PON2融合蛋白在大肠杆菌中进行表达,以HIS-PON2作为免疫原制备鼠mAb,以GST-PON2作为筛选抗原。采用Western blot、间接免疫荧光鉴定mAb的特异性。结果:GST-PON2和HIS-PON2融合蛋白均在大肠杆菌中获得高效表达,经常规的细胞融合和筛选获得2株可稳定分泌抗PON2的杂交瘤细胞株,这2株抗体可以识别HepG2细胞中的靶蛋白。结论:成功制备出2株抗PON2的mAb,并通过免疫荧光技术检测了该蛋白在HepG2细胞中的分布,为进一步进行PON2蛋白的的研究提供了有效的工具。
Objective: To prepare monoclonal antibody (mAb) for PON2 (paraxonase2) and preliminary identification. Methods: The human PON2 protein sequence was analyzed by bioinformatics method. The fragments with low homology and high immunogenicity and hydrophilicity were selected to construct recombinant plasmid pGEX-4T-1-PON2 and PET- The 32a-PON2, GST-PON2 and HIS-PON2 fusion proteins were expressed in E. coli. The murine mAbs were prepared using HIS-PON2 as the immunogen and GST-PON2 as the screening antigen. The specificity of mAb was identified by Western blot and indirect immunofluorescence. RESULTS: Two fusion proteins of GST-PON2 and HIS-PON2 were highly expressed in E. coli. Two hybridoma cell lines stably secreting anti-PON2 were obtained by conventional cell fusion and screening. These two antibodies could recognize HepG2 cells Of the target protein. CONCLUSION: Two mAbs against PON2 were successfully prepared and the distribution of the mAb in HepG2 cells was detected by immunofluorescence assay, which provided an effective tool for the further study of PON2 protein.