磷酸酪氨酸蛋白专一抗体的制备和纯化

Preparation and Purification of O Phosphotyrosine Protein Specific Antibody

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．自抗血清中分离获得抗体．自酪胺合成磷酸酪胺，并偶联到溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ上．抗体经磷酸酪胺－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化，所得抗体专一性强，Ｄｏｔｂｌｏｔ显示：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，也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．

Rabbits were immunized with bovine serum albumin(BSA)which was coupled to phosphorylated tyrosine to produce antiserum.Antibodies were isolated from the serum.O Phosphotyramine was synthesized from tyramine and purified and then coupled to BrCN activated Sepharose 4B.Specific antibodies were purified further by affinity chromatography with O Phosphotyramine Sepharose 4B.Specificity of the purified multiclonal antibodies was assessed by dot blot and found to be very high.They specifically reacted with tyrosine phosphorylated proteins including modified BSA,lysozyme,ovalbumin to which O Phosphotyrosine were attached through the reaction of EDC,but did not react with normal forms of lysozyme,ovalbumin without phosphorylated tyrosine residues;Neither did they recognize BSA the major skeleton of original antigen nor react with modified ovalbumin with serine or threonine residues phosphorylated.