尖吻蝮蛇蛇毒多克隆抗体纯化及生物活性研究  

Purification and biological activity of polyclonal antibodies against Deinagkistrodon acutus venoms

作　　者：曹宇亮[1,2] 张雷[1] 刘明华[1] 

机构地区：[1]第三军医大学西南医院急救部,重庆400038 [2]解放军第98医院急诊科,浙江湖州313000

出　　处：《第三军医大学学报》2014年第9期932-936,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(81071537);重庆市科技攻关计划项目(CSTC2010AC5026);第三军医大学临床创新基金(2009XLC15)~~

摘　　要：目的通过实验纯化出特异性尖吻蝮蛇蛇毒多克隆抗体并检测其生物活性。方法以尖吻蝮蛇蛇毒免疫新西兰兔,利用Econo-Pac Protein A kit及CNBr activated sepharose 4B凝胶对兔源性抗蛇毒血清抗体进行纯化,制备特异性尖吻蝮蛇蛇毒多克隆抗体,经Western blot和ELISA方法对抗体进行评价,通过出血抑制实验及致死性保护实验评价抗体的生物活性。结果成功纯化出特异性抗尖吻蝮蛇蛇毒多克隆抗体,ELISA检测抗体效价>3 200,Western blot证实多克隆抗体能识别蛇毒中大多数毒素蛋白,出血抑制实验证实该抗体能有效抑制蛇毒所致的皮下出血,在致死性保护实验中,该抗体对2LD50和4LD50蛇毒分别起到100%和54%的保护作用。结论纯化获得的尖吻蝮蛇蛇毒多克隆抗体具有较好特异性和生物活性。Objective To purify specific polyclonal antibodies against Deinagkistrodon acutus venoms for new antivenom research. Methods New Zealand white rabbits were immunized with Deinagkistrodon acutus venom. The IgG fraction was purified by affinity chromatography with the Econo-Pac Protein A Kit. Specific antibodies were purified by immuno-affinity chromatography with CNBr activated sepharose 4B.Then the polyclonal antibodies were tested by ELISA and Western blotting to evaluate its immunocompetence. Antibody biological activity of the polyclonal antibodies was evaluated by lethality neutralization assay and neutralization of venom hemorrhagic activity. Results Through this way, we successfully prepared specific polyclonal antibodies against Deinagkistrodon acutus venoms. ELISA showed the antibody titers higher than 3 200. Western blotting indicated that the polyclonal antibodies specifically recognized the most protein of Deinagkistrodon acutus venoms. Neutralization of venom hemorrhagic activity demonstrated the polyclonal antibodies effectively neutralized subcutaneous hemorrhage induced by Deinagkistrodon acutus venoms. In the lethality neutralization assay, the polyclone antibodies provided 100% and 54% protection against 2LD50 and 4LD50 dose Deinagkistrodon acutus venoms respectively. Conclusion The specific polyclonal antibodies against Deinagkistrodon acutus are purified successfully with good specificity and bio-activity.

关 键 词：尖吻蝮蛇 多克隆抗体 抗体纯化 

分 类 号：R392.7[医药卫生—免疫学] R945[医药卫生—基础医学]