基于抗原表位的抗尖吻蝮蛇毒磷脂酶A2抗体制备  

Preparation of Antibody of Phospholipase A2 from Deinagkistrodon acutus Vemon Based on Antigenic Epitope

作　　者：朱海婷 李恒[2] 杨万舟 张文浩 余晓东[1] 和七一[1] ZHU Haiting;LI Heng;YANG Wanzhou;ZHANG Wenhao;YU Xiaodong;HE Qiyi(Chongqing Engineering Research Center of Bioactive Substance,Ministry of Education Engineering Research Center of Active Substance and Biotechnology,College of Life Sciences,Chongqing Normal University,Chongqing 401331;Chongqing Public Security Bureau Evidence Identification Center,Chongqing 400021,China)

机构地区：[1]重庆师范大学生命科学学院、教育部活性物质生物技术工程研究中心、重庆市生物活性物质工程研究中心,重庆401331 [2]重庆市公安局证据鉴定中心,重庆400021

出　　处：《重庆师范大学学报(自然科学版)》2024年第6期54-61,共8页Journal of Chongqing Normal University:Natural Science

基　　金：国家自然科学基金面上项目(No.3227030453);重庆市自然科学基金面上项目(No.CSTB2022NSCQ-MSX0423)。

摘　　要：为探究尖吻蝮(Deinagkistrodon acutus)蛇毒磷脂酶A2(phospholipase A2,PLA2)的抗原表位并制备针对该酶的特异性抗体,通过IEDB-Bepipred 2.0在线服务器对PLA2的氨基酸序列进行线性B细胞抗原表位预测,确定抗原位点氨基酸序列,并将它们连接到表达载体pET-28a(+)中进行原核表达获得重组蛋白。将纯化后的重组蛋白用于小鼠(Mus musculus)免疫以制备针对尖吻蝮蛇PLA2的抗体。通过酶联免疫吸附试验(ELISA)和蛋白质印迹法(Western blot)检测分析小鼠血清中的抗体情况,评价抗体对尖吻蝮蛇毒PLA2活性的抑制作用。成功预测了尖吻蝮蛇毒PLA2的6个抗原位点氨基酸序列,并表达了相应的重组蛋白,该蛋白的相对分子质量约为16.5 kDa。ELISA和Western blot检测分析结果表明小鼠血清对尖吻蝮蛇毒PLA2的抗体滴度高于1∶256,并且与尖吻蝮蛇毒中相对分子质量为14~16 kDa的PLA2蛋白产生强烈的结合反应。体外酶活测定实验结果表明10μL重组蛋白免疫血清使6μg尖吻蝮蛇毒PLA2活性由100%降低至62.2%。研究结果提示基于尖吻蝮蛇毒PLA2设计的抗原表位能够有效刺激小鼠产生免疫应答反应,并成功制备出可抑制尖吻蝮蛇毒PLA2活性的血清抗体。To investigate the antigenic epitopes of phospholipase A2(PLA2)from Deinagkistrodon acutus venom and to prepare specific antibodies against this enzyme,the amino acid sequence of PLA2 was analyzed using the IEDB-Bepipred 2.0 online server for linear B-cell epitope prediction.The identified antigenic epitopes were cloned into the expression vector pET-28a(+)for prokaryotic expression,and the resulting recombinant protein was purified.The purified recombinant protein was used to immunize mice(Mus musculus)for antibody production.Enzyme-linked immunosorbent assay(ELISA)and Western blot were employed to evaluate the antibody titers in mouse sera and their binding activity against PLA2 from D.acutus venom,as well as to assess the inhibitory effect of the antibodies on PLA2 enzymatic activity.Six antigenic epitope sequences of PLA2 were successfully predicted,and the corresponding recombinant protein,with a relative molecular weight of approximately 16.5 kDa,was expressed.ELISA and Western blot analyses showed that mouse sera exhibited antibody titers exceeding 1∶256 and strongly bound to PLA2 proteins with relative molecular weights of 14~16 kDa in D.acutus venom.In vitro enzymatic activity assays demonstrated that 10μL of immunized serum reduced the activity of 6μg PLA2 from 100%to 62.2%.These findings indicate that the antigenic epitopes designed based on D.acutus venom PLA2 effectively stimulated an immune response in mice,resulting in the successful preparation of serum antibodies capable of neutralizing PLA2 from D.acutus venom.

关 键 词：磷脂酶A2 尖吻蝮 B细胞抗原表位 原核表达 

分 类 号：Q556[生物学—生物化学] Q786