抗小鼠SHP-1、SHP-2和SHIP三种蛋白酪氨酸磷酸酶单克隆抗体的制备及鉴定

为了制备抗小鼠蛋白酪氨酸磷酸酶的单克隆抗体(mAb),分别以SHP-1、SHP-2和SHIP重组蛋白为抗原免疫BALB/c小鼠,通过B淋巴细胞杂交瘤技术制备抗相应磷酸酶的mAb。用Western blot检测mAb对重组蛋白和细胞中天然磷酸酶的反应性。共获得12株可稳定分泌抗磷酸酶mAb的杂交瘤细胞株。其中6株可分泌抗SHP-1 mAb(LX-SHP1.1~LX-SHP1.6),3株可分泌抗SHP-2 mAb(LX-SHP2.1~LX-SHP2.3),3株分泌抗SHIP mAb(LX-SHIP1~LX-SHIP3)。LX-SHP1.2~LX-SHP1.6,LX-SHP2.1和LX-SHP2.3,以及LX-SHIP2和LX-SHIP3可应用于相应重组蛋白的Westernblot检测。LX-SHP1.5,LX-SHP2.3,以及LX-SHIP2在EL-4细胞及原代T细胞相应天然磷酸酶分子的Western blot检测中有较好的实验效果。

Preparation and identification of monoclonal antibodies against mouse tyrosine phosphatases SHP-1, SHP-2 and SHIP

The monoclonal antibodies(mAb) against mouse tyrosine phosphatases SHP-1,SHP-2 and SHIP were prepared by using the respective recombinant proteins as immunogens to immunize BALB/c mice,and the B lymphocyte hybridization technique to prepare the corresponding mAb.Western blot assay was used to detect the reactivity of the mAb with recombinant proteins and the natural cellular phosphatases.Twelve hybridoma cell lines secreting mAb against SHP-1,SHP-2 and SHIP were thus obtained,among which 6 strains(LX-SHP-1.1~LX-SHP-1.6)secreting anti-SHP-1 antibodies,3 strains(LX-SHP2.1~LX-SHP2.3) secreting anti-SHP-2 antibodies,and 3 strains(LX-SHP-1~LX-SHIP-3) secreting anti-SHIP antibodies.In the Western blot assay,LX-SHP1.2~SHP(1.6),LX-SHP(2.1),LX-SHP(2.3),LX-SHIP-2 and LX-SHIP-3 could be used for the detection of the respective recombinant proteins,whereas,LX-SHP1.5,LX-SHP2.3 and LX-SHIP-2 showed better results for the detection of the natural phosphatases when the EL-4 murine thymus lymphoma cells and the primary T cell lysates were employed.These results would lay the foundation for the studies in immunoreceptor-mediated signal transduction and the regulation of the phosphatase activities.