抗磷酸酪氨酸抗体的制备、纯化和应用

Preparation, purification and application of O phosphotyrosine protein specific antibody

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)

Objective To prepare the O-phosphotyrosine protein specific antibody and by using it to study the effects of ischemia/reperfusion on the immunoreactivity of phosphotyrosine protein(p-tyr-pr) in gerbil hippocampus.?Methods Rabbits were immunized with bovine serum albumin (BSA) which was coupled to phosphorylated tyrosine to produce the antiserum. The antibodies were first salted from the serum and the antibodies were purified further by affinity chromatography using a self-prepared O-phosphotyrosine-sepharose 4B column. The specificity of the purified polyclonal antibodies was assessed by dot blot and found to be very high.?Results They specifically reacted only with tyrosine-phosphorylated proteins, including modified BSA, lysozyme and ovalbumin, they did not react with normal forms of lysozyme, ovalbumin having no phosphorylated tyrosine residues, nor did they recognize BSA(the major skeleton of original antigen).They did not react with modified ovalbumin with serine or threonine residues phosphorylated either. At 6 h of reperfusion, a marked increase in p-tyr-pr immunoreactivity was seen in the hippocampus compared with the control. The increase in p-tyr-pr immunoreactivity induced by brain ischemia/reperfusion was partially antagonized by ketamine and nifedipine administered prior to cerebral ischemia.?Conclusions The affinity-purified polyclonal rabbit anti-p-tyr-pr antibody is highly specific for p-tyr-pr. The p-tyr-pr immunoreactivity increase in the hippocampus induced by brain ischemia/reperfusion is related to N-methyl-D-aspartate receptor channel and L-type voltage gated calcium channel.