长白白眉蝮蛇蛇毒纤溶酶原激活剂单克隆抗体的制备及性质研究
发布时间：2018-08-27 07:12
【摘要】： 本课题以长白白眉蝮蛇蛇毒为原料，通过DEAE-Sephadex A-50离子交换层析、Sephadex G-75凝胶层析、Lysine Sepharose TM4B层析得到蛇毒纤溶酶原激活剂。以此为抗原对BALB／C小鼠进行免疫，通过杂交瘤技术获得免疫BALB／C小鼠脾细胞和小鼠骨髓瘤细胞的阳性融合细胞株，使用定量的阳性融合细胞诱导小鼠产生效价高的腹水，将小鼠腹水用辛酸．硫酸铵法进行一系列的纯化。共获得4株长白白眉蝮蛇蛇毒纤溶酶原激活剂单克隆抗体，为287、3C11、4E7、4H10。进行较系统的免疫特性的鉴定：4株单抗特异性高，与u-PA、BSA均无交叉反应；腹水效价8200～10000；免疫球蛋白亚类为IgG1和IgG2a；4株抗体中，287和3C11可明显抑制长白白眉蝮蛇蛇毒纤溶酶原激活剂活性，而4E7、4H10则对其活性无明显影响。287和3C11这2株抗体为建立ELISA法检测长白白眉蝮蛇蛇毒纤溶酶原激活剂含量以及研究其功能和结构的关系奠定了基础，，并为该成分的亲和层析提供重要配体。
[Abstract]:In this study, the venom plasminogen activator of snake venom was obtained by DEAE-Sephadex A-50 ion exchange chromatography Sephadex G-75 gel chromatography and Lysine Sepharose TM4B chromatography using Agkistrodon halys venom as raw material. The positive fusion cell lines of spleen cells and myeloma cells of BALB/C mice were obtained by hybridoma technique. The positive fusion cells were induced to produce ascites with high titer by using quantitative positive fusion cells. Mouse ascites were purified by octanoic acid and ammonium sulfate method. Four monoclonal antibodies against plasminogen activator of Agkistrodon acutus venom were obtained. The specific of the McAbs was high, and there was no cross reaction with u-PABSA. The ascites titer was 8200,10000. the immunoglobulin subclasses were IgG1 and IgG2a;. The activity of plasminogen activator in venom of Agkistrodon halys was significantly inhibited by 3C11. However, 4E7H _ (10) had no significant effect on the activity of. 287 and 3C11, which laid a foundation for the detection of plasminogen activator in Agkistrodon halys venom by ELISA, and for the study of the relationship between the function and structure of plasminogen activator of Agkistrodon halys venom. It also provides an important ligand for affinity chromatography.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2206511
[Abstract]:In this study, the venom plasminogen activator of snake venom was obtained by DEAE-Sephadex A-50 ion exchange chromatography Sephadex G-75 gel chromatography and Lysine Sepharose TM4B chromatography using Agkistrodon halys venom as raw material. The positive fusion cell lines of spleen cells and myeloma cells of BALB/C mice were obtained by hybridoma technique. The positive fusion cells were induced to produce ascites with high titer by using quantitative positive fusion cells. Mouse ascites were purified by octanoic acid and ammonium sulfate method. Four monoclonal antibodies against plasminogen activator of Agkistrodon acutus venom were obtained. The specific of the McAbs was high, and there was no cross reaction with u-PABSA. The ascites titer was 8200,10000. the immunoglobulin subclasses were IgG1 and IgG2a;. The activity of plasminogen activator in venom of Agkistrodon halys was significantly inhibited by 3C11. However, 4E7H _ (10) had no significant effect on the activity of. 287 and 3C11, which laid a foundation for the detection of plasminogen activator in Agkistrodon halys venom by ELISA, and for the study of the relationship between the function and structure of plasminogen activator of Agkistrodon halys venom. It also provides an important ligand for affinity chromatography.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
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