瓜氨酸化α-烯醇化酶特异性单克隆抗体制备及其在阿尔茨海默病动物模型中的检测应用

发布时间：2018-08-17 11:21

【摘要】：本研究利用杂交瘤技术制备瓜氨酸化α-烯醇化酶特异性单克隆抗体,并探究瓜氨酸化α-烯醇化酶抗体在检测阿尔茨海默病(AD)动物模型中的作用。利用生物信息学软件分析预测α-烯醇化酶的B细胞优势表位,选取~9R-~(25)F位氨基酸序列,将该表位多肽中2个精氨酸替换为瓜氨酸,化学合成瓜氨酸化α-烯醇化酶~9R-~(25)F多肽(citrullinatedα-enolase peptide,CEP)。采取活泼酯法将CEP多肽与BSA偶联制备CEP-BSA偶联抗原并免疫BALB/c小鼠,杂交瘤技术制备CEP单克隆抗体。免疫组织化学和Western blotting对其特异性进行验证。CEP-BSA偶联抗原免疫小鼠能诱发特异性抗体反应,经细胞融合、筛选得到一株稳定分泌CEP特异性单克隆抗体的杂交瘤细胞,命名为9B5。Western blotting分析显示该单克隆抗体能特异性结合瓜氨酸化α-烯醇化酶,免疫组织化学检测显示9B5单抗能结合AD模型大鼠脑组织细胞上瓜氨酸化的α-烯醇化酶。本研究成功制备的瓜氨酸化α-烯醇化酶特异性单克隆抗体将有助于进一步研究瓜氨酸化α-烯醇化酶在相关疾病发病中的作用。
[Abstract]:In this study, we used hybridoma technique to prepare specific monoclonal antibodies against citrullated 伪 -enolase, and to explore the role of citrullated 伪 -enolase antibody in the detection of Alzheimer's disease (AD) animal model. The B cell dominant epitopes of 伪 -enolase were predicted by bioinformatics software. The amino acid sequence of 9R-25 F site was selected, two arginine of the peptide were replaced with citrullinic acid, and the citrullinated 伪 -enolase 9R-25 F peptide (CEP) was chemically synthesized. CEP peptide was conjugated with BSA to prepare CEP-BSA conjugated antigen and immunized BALB/c mice by active ester method. Monoclonal antibody to CEP was prepared by hybridoma technique. Immunohistochemistry and Western blotting showed that CEP-BSA conjugated antigen could induce specific antibody reaction in mice. A hybridoma cell line secreting CEP monoclonal antibody stably was obtained by cell fusion. 9B5.Western blotting analysis showed that the monoclonal antibody could specifically bind to 伪 -enolase, and 9B5 monoclonal antibody could bind to 伪 -enolase in brain cells of AD model rats. The successful preparation of specific monoclonal antibodies against melamine acidated 伪 -enolase will be helpful to further study the role of citrullated 伪 -enolase in the pathogenesis of related diseases.
【作者单位】：华中师范大学生命科学学院湖北省遗传调控与整合生物学重点实验室;
【基金】：湖北省自然科学基金重点项目(2014CFA079) 中央高校基本科研业务费项目资助(CCNU16A02044)
【分类号】：R-332;R749.16

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