抗尖吻蝮蛇蛇毒金属酶类共同基序IgY抗体的制备与研究

目的]制备针对尖吻蝮蛇蛇毒金属蛋白酶类共同基序的IgY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IgY抗体.采用ELISA和Western blot等方法对IgY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IgY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IgY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 IgY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IgY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.

IgY against Bleeding Snake Venom Metalloproteinase Consensus Sequence,Preparation and Research

Objective]To prepare and research the anti-snake venom activity of the Ig Y antibody, which was specific against the consensus sequence of Deinagkistrodon metalloproteinases. Methods]Design and synthesis of two antigen peptide PT1 and PT2, were highly conserved and closely correlated with the enzyme activity. The two peptides were covalent with keyhole limpet hemocyanin（KLH） to form antigen compound KLH-PT1 and KLH-PT2,and then hens were immunized to obtain Ig Y antibody. Double immunodiffusion（DID）, ELISA essay, Western blot technology were carried out to systemically research the valence and cross-reaction property of the Ig Y with the Deinagkistrodon and Gloydius brevicaudus＇ venom. Finally, mice subcutaneous bleeding toxic model was adopted to evaluate the neutralization metalloproteinase active capacity of the Ig Y. Results]ELISA and Western blot indicated that, after three times of immune, high titer Ig Y against corresponding antigens was obtained; amount which, the Ig Y against total Deinagkistrodon venom and KLH-PT1 can cross-react with the Deinagkistrodon venom and Gloydius brevicaudus venom, and the former was superior to the later. It was unexpected that the Ig Y against KLH-PT2 can not react with both venoms in vitro, but can strongly neutralize the toxicity of bleeding caused by venom metalloproteinase on mice, which was remarkably active than the two others. This is probably correlated with the conformation change of the venom metalloproteinase in vivo; more evidences have to be made. Conclusion]This research confirmed that it was possible to obtain Ig Y, which could cross-react with different blood venom through design the consensus antigen sequence of the venom metalloproteinase. And it would be an alternative way to produce high efficiency, low toxicity antiserum.