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[目的]制备针对尖吻蝮蛇蛇毒金属蛋白酶类共同基序的Ig Y抗体,并对其中和蛇毒活性的作用进行研究。[方法]通过分析尖吻蝮蛇蛇毒各类金属蛋白酶类共有序列,设计合成与其酶活性密切相关且高度保守的抗原肽PT1和PT2;偶联复合物KLH-PT1、KLH-PT2免疫母鸡获得Ig Y抗体。采用ELISA和Western blot等方法对Ig Y效价及与尖吻蝮蛇蛇毒和短尾蝮蛇蛇毒的交叉反应特性进行初步研究;最后通过抗小鼠皮下出血实验对Ig Y中和活性进行评价。[结果]ELISA、Western blot结果表明,抗KLH-PT1、抗尖吻蝮蛇蛇毒Ig Y均可与尖吻蝮蛇蛇毒、短尾蝮蛇蛇毒发生交叉反应且后者显著强于前者;抗KLH-PT2 Ig Y在体外与尖吻蝮蛇蛇毒、短尾蝮蛇蛇毒无明显交叉反应,在体内抗出血实验中却表现出很强的中和毒性作用,并且显著优于前两者。[结论]通过设计金属蛋白酶共有序列抗原肽,制备了能与多种血循型蛇毒交叉反应的Ig Y抗体,这为制备特异、高效、低毒性且广谱的抗出血型蛇毒抗体奠定了基础。
[Objective] The research aimed to prepare the Ig Y antibody directed against the metalloprotease common motif of Agkistrodon versicolor, and study its effect on neutralizing the activity of snake venom. [Methods] By analyzing the consensus sequences of various metalloproteinases of Agkistrodon versicolor, we designed and synthesized the highly conserved antigenic peptides PT1 and PT2, which were closely related to their enzymatic activities. The conjugated complexes KLH-PT1 and KLH-PT2 were used to immunize hens Ig Y antibody. The IgY titer and the cross-reactivity with Agkistrodon halyae snake venom and snakehead snake venom were studied by ELISA and Western blot. Finally, Ig Y neutralization activity was evaluated by anti-mouse subcutaneous hemorrhage. [Result] The results of ELISA and Western blot showed that anti-KLH-PT1 and IgY of Agkistrodon halysii could cross-react with Agkistrodon halysii and Snakehead snake venom and the latter were significantly stronger than the former; anti-KLH -PT2 Ig Y had no significant cross-reaction with Agkistrodon acutus venom and snake venom in vitro, but showed a strong neutralization toxicity in anti-hemorrhage in vivo and was significantly better than the former two. [Conclusion] The Ig Y antibody that can cross-react with various venomous snake venom was prepared by designing the metalloproteinase consensus peptide, which lays the foundation for the preparation of a specific, efficient and low toxicity broad-spectrum anti-hemorrhagic snake venom antibody .