4-戊基苯酚单克隆抗体的制备及其可变区的同源建模

发布时间：2018-05-16 22:00

本文选题：-戊基苯酚(-AP) + 单克隆抗体　；参考：《细胞与分子免疫学杂志》2017年03期

【摘要】：目的制备4-戊基苯酚(4-AP)单克隆抗体(mAb),通过分子生物学方法对其可变区cDNA克隆和同源建模,为4-AP单链抗体制备及抗体分子层面改造奠定基础,同时为4-AP的免疫学检测提供一种新方法。方法将免疫小鼠脾脏细胞与Sp2/0细胞融合,筛选出F10阳性单抗细胞株。通过其mRNA的抽提,克隆出可变区cDNA序列,利用生物软件进行同源建模和分子对接。结果在最适条件下,其间接竞争ELISA(ic-ELISA)公式为y=A_2+(A_1-A_2)/(1+(x/x_0)~p)(A_1=1.28;A_2=-0.07;x_0=12 560.75;p=0.74),相关系数(R~2)为0.997,其最低检测限为0.65μg/mL。mAb重链和轻链分别属于IgG1型和Kappa型,其与4-AP的结合力为氢键和疏水作用。结论成功制备出1株稳定分泌的4-AP mAb的杂交瘤细胞株。通过同源建模对抗体可变区空间结构进行深入研究,为单链抗体及抗体改造提供参考,同时为4-AP含量检测提供新方法。
[Abstract]:Objective to prepare the monoclonal antibody of 4-pentylphenol 4-APs and clone its variable region cDNA by molecular biology, and to establish the foundation for the preparation of 4-AP single chain antibody and the modification of its molecular level. It also provides a new method for immunological detection of 4-AP. Methods spleen cells of immunized mice were fused with Sp2/0 cells and F 10 positive McAb cell lines were screened. Through the extraction of its mRNA, the variable region cDNA sequence was cloned, and the homologous modeling and molecular docking were carried out by using biological software. Results under the optimum conditions, the indirect competition ELISA-ELISA-ELISA-ELISAA) formula is y=A_2 / A1-A / 2 / T / 1 / T / 1 / X / 0 / T / 1 / 1 / 0 / 0 / 1 / 1 / 1 / 1 / 0 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 0 / 0 / 0 / 0 / 0 / 12 560.75p0.74. the minimum detection limit is 0.997. the minimum detection limit is 0.65 渭 g/mL.mAb heavy chain and Kappa type, respectively, and its binding force with 4-AP is hydrogen bond and hydrophobic interaction. The results show that the maximum detection limit is 0.65 渭 g/mL.mAb heavy chain and Kappa type respectively, and its binding force with 4-AP is hydrogen bond and hydrophobicity. Conclusion A hybridoma cell line with stable secretion of 4-AP mAb was successfully prepared. The spatial structure of variable region of antibody was studied by homology modeling, which provided a reference for scFV and antibody modification, and a new method for 4-AP content detection.
【作者单位】：华东理工大学生物反应器工程国家重点实验室;上海出入境检测检验局工业产品和原材料检测中心;
【基金】：上海检验检疫局科技计划(Hk016-2014)
【分类号】：R392

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