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目的 制备抗磷酸酪氨酸多克隆抗体 ,利用此抗体研究缺血再灌注对海马磷酸酪氨酸蛋白 (p -tyr -pr)免疫反应性的影响。方法 以偶联磷酸化酪氨酸的牛血清白蛋白 (BSA)作为免疫原免疫兔获得抗血清。将磷酸酪氨酸偶联到溴化氰活化的sepharose 4B上。抗血清经磷酸酪氨酸 -sepharose 4B亲和柱纯化 ,所得抗体专一性强 ,并经dotblot鉴定。结果 抗体仅对酪氨酸磷酸化的蛋白质包括酪氨酸磷酸化的血清白蛋白、溶菌酶、卵清蛋白起抗原抗体反应 ,而不识别非酪氨酸磷酸化的溶菌酶、卵清蛋白 ;不识别作为免疫原的骨架成分BSA ,也不识别丝氨酸磷酸化的卵清蛋白和苏氨酸磷酸化的卵清蛋白 ;缺血 /再灌注 6h海马各区p -ty -pr免疫反应性明显增强 ,氯胺酮或硝苯吡啶对此有拮抗作用。结论 亲和纯化后的抗体是高度特异性的。缺血 /再灌注后p -tyr-pr免疫反应性增强与N -甲基 -D -天冬氨酸 (NMDA)受体以及电压门控的钙离子通道有关
Objective To prepare anti-phosphotyrosine polyclonal antibody to study the effect of ischemia-reperfusion on the immunoreactivity of hippocampal phosphotyrosine protein (p-tyr-pr). Methods Anti-serum was obtained by immunizing rabbits with bovine serum albumin conjugated with phosphotyrosine (BSA) as immunogen. Phosphotyrosine was coupled to cyanogen bromide-activated sepharose 4B. The antiserum was purified by phosphotyrosine-sepharose 4B affinity column and the resulting antibody was specific and identified by dot blot. Results The antibodies only reacted with tyrosine phosphorylated proteins, including tyrosine phosphorylated serum albumin, lysozyme and ovalbumin, but not non-tyrosine phosphorylated lysozyme and ovalbumin. It did not recognize BSA as a scaffold of immunogen, nor did it recognize serine-phosphorylated ovalbumin and threonine-phosphorylated ovalbumin. Immunoreactivity of p -ty-pr in hippocampus was significantly increased at 6 h after ischemia / reperfusion, Ketamine or nifedipine antagonized this effect. Conclusion The affinity-purified antibodies are highly specific. Increased p-tyr-pr immunoreactivity after ischemia / reperfusion is associated with N-methyl-D-aspartate (NMDA) receptors and voltage-gated calcium channels