小鼠抗NLRP3单克隆抗体的制备及鉴定
Preparation and assessment of a monoclonal antibody against mouse NLRP3
姚羽慧 1崔蒙蒙 2孟广勋2
作者信息
- 1. 苏州大学,苏州医学院,巴斯德学院,江苏苏州 215000
- 2. 中国科学院上海免疫与感染研究所,微生物、发育与健康研究中心,中国科学院分子病毒与免疫重点实验室,上海 200031
- 折叠
摘要
目的 制备小鼠抗含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)的单克隆抗体(mAb)并检测其特异性.方法 将编码小鼠NLRP3基因外显子3(Ms-N3)的基因片段插入载体p36-G3-throhFc中构建重组质粒Ms-N3-throhFc,然后将该质粒转染到HEK293F细胞中,进行真核蛋白表达.进一步利用蛋白A亲和层析柱纯化得到Ms-N3蛋白并免疫NLRP3基因敲除(NLRP3-/-)小鼠,将免疫小鼠的脾细胞与SP2/0骨髓瘤细胞融合形成杂交瘤细胞.进而通过ELISA和免疫荧光方法筛选能够分泌特异性识别小鼠NLRP3的mAb的杂交瘤细胞.结果 成功构建Ms-N3-throhFc重组质粒并证明该质粒能在HEK293F细胞中稳定表达.ELISA初步筛选获得12株杂交瘤细胞,经过免疫荧光实验检测发现其中的9-B8-3-2-C5分泌的mAb能够特异性识别非变性的小鼠NLRP3蛋白,该mAb的重链亚型为IgM,轻链亚型为κ.结论 成功获得小鼠抗NLRP3 mAb.
Abstract
Objective To prepare a monoclonal antibody(mAb)against mouse NOD-like receptor family pyrin domain-containing 3(NLRP3)and assess its specificity.Methods A gene fragment encoding mouse NLRP3 exon3(Ms-N3)was inserted into the vector p36-G3-throhFc to construct a recombinant plasmid named Ms-N3-throhFc.This plasmid was then transfected into HEK293F cells for eukaryotic expression.NLRP3-/-mice were immunized with Ms-N3 protein purified using a protein A chromatography column,and splenocytes from the immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cells.Specific mAbs against murine NLRP3 from hybridoma cells were screened using ELISA and immunofluorescence assay(IFA).Results The Ms-N3-throhFc recombinant plasmid was successfully constructed and exhibited stable expression in HEK293F cells.Twelve hybridoma cell lines were initially screened using ELISA.IFA revealed that the mAb secreted by the 9-β8-3-2-C5 cell line specifically recognized the native form of mouse NLRP3 protein.The heavy and light chain subtypes of this mAb were identified as IgM and κ,respectively.Conclusion A monoclonal antibody against mouse NLRP3 has been successfully prepared.
关键词
含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)/真核表达/蛋白纯化/单克隆抗体(mAb)/免疫荧光Key words
NLRP3/eukaryotic expression/protein purification/monoclonal antibody(mAb)/immunofluorescence引用本文复制引用
出版年
2024
NETL
NSTL
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