瓜氨酸化α-烯醇化酶特異性單克隆抗體制備及其在阿爾茨海默病動物模型中的檢測應用
發(fā)布時間：2018-08-17 11:21
【摘要】：本研究利用雜交瘤技術(shù)制備瓜氨酸化α-烯醇化酶特異性單克隆抗體,并探究瓜氨酸化α-烯醇化酶抗體在檢測阿爾茨海默病(AD)動物模型中的作用。利用生物信息學軟件分析預測α-烯醇化酶的B細胞優(yōu)勢表位,選取~9R-~(25)F位氨基酸序列,將該表位多肽中2個精氨酸替換為瓜氨酸,化學合成瓜氨酸化α-烯醇化酶~9R-~(25)F多肽(citrullinatedα-enolase peptide,CEP)。采取活潑酯法將CEP多肽與BSA偶聯(lián)制備CEP-BSA偶聯(lián)抗原并免疫BALB/c小鼠,雜交瘤技術(shù)制備CEP單克隆抗體。免疫組織化學和Western blotting對其特異性進行驗證。CEP-BSA偶聯(lián)抗原免疫小鼠能誘發(fā)特異性抗體反應,經(jīng)細胞融合、篩選得到一株穩(wěn)定分泌CEP特異性單克隆抗體的雜交瘤細胞,命名為9B5。Western blotting分析顯示該單克隆抗體能特異性結(jié)合瓜氨酸化α-烯醇化酶,免疫組織化學檢測顯示9B5單抗能結(jié)合AD模型大鼠腦組織細胞上瓜氨酸化的α-烯醇化酶。本研究成功制備的瓜氨酸化α-烯醇化酶特異性單克隆抗體將有助于進一步研究瓜氨酸化α-烯醇化酶在相關(guān)疾病發(fā)病中的作用。
[Abstract]:In this study, we used hybridoma technique to prepare specific monoclonal antibodies against citrullated 偽 -enolase, and to explore the role of citrullated 偽 -enolase antibody in the detection of Alzheimer's disease (AD) animal model. The B cell dominant epitopes of 偽 -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 偽 -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 偽 -enolase, and 9B5 monoclonal antibody could bind to 偽 -enolase in brain cells of AD model rats. The successful preparation of specific monoclonal antibodies against melamine acidated 偽 -enolase will be helpful to further study the role of citrullated 偽 -enolase in the pathogenesis of related diseases.
【作者單位】： 華中師范大學生命科學學院湖北省遺傳調(diào)控與整合生物學重點實驗室;
【基金】：湖北省自然科學基金重點項目(2014CFA079) 中央高�；究蒲袠I(yè)務費項目資助(CCNU16A02044)
【分類號】：R-332;R749.16
[Abstract]:In this study, we used hybridoma technique to prepare specific monoclonal antibodies against citrullated 偽 -enolase, and to explore the role of citrullated 偽 -enolase antibody in the detection of Alzheimer's disease (AD) animal model. The B cell dominant epitopes of 偽 -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 偽 -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 偽 -enolase, and 9B5 monoclonal antibody could bind to 偽 -enolase in brain cells of AD model rats. The successful preparation of specific monoclonal antibodies against melamine acidated 偽 -enolase will be helpful to further study the role of citrullated 偽 -enolase in the pathogenesis of related diseases.
【作者單位】： 華中師范大學生命科學學院湖北省遺傳調(diào)控與整合生物學重點實驗室;
【基金】：湖北省自然科學基金重點項目(2014CFA079) 中央高�；究蒲袠I(yè)務費項目資助(CCNU16A02044)
【分類號】：R-332;R749.16
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