【摘要】 将小鼠凝血因子Ⅸ (mFⅨ )cDNA蛋白编码区序列克隆至原核表达载体 pQE30 ,在大肠杆菌M 15中获得高效表达 (约占细菌蛋白总量的 5 1 2 % ) ,并经过SDS PAGE纯化获得均一的重组mFⅨ蛋白 .以此蛋白为抗原 ,免疫Lou/MN系大鼠 ,取其脾细胞与小鼠骨髓瘤细胞 (Sp 2 / 0 )融合后 ,经 3次克隆化选择获 2株杂交瘤细胞(H4B5和C2B10 ) .以 1× 10 6杂交瘤细胞注射免疫缺陷型裸鼠 ,10d后获得腹水 .对此单抗的功能和特异性初步研究表明 ,所制备的抗小鼠FⅨ单抗可用于Western免疫印迹等研究 .更多还原

【Abstract】 The coding region of mouse coagulation factor Ⅸ (mFⅨ) cDNA was subcloned into the pQE30 plasmid. High level expression of mFⅨ polypeptide (51.2% in total bacterial proteins) in Ecoli M15 was purified by SDS PAGE. Hybridoma was derived by fusion spleen cells of Lou/MN strain rat immunized with recombinant mFⅨ(rmFⅨ) and mouse myeloma Sp2/0. The cloned positive hybridoma cells (1×10 6 mL -1 ) was injected into abdomen of nude mice and the monoclonal antibody (mAb) to mFⅨ was harvested 10 days later. The systematic screening of its function and specificity suggested that this anti mouse mAb could be employed for utilization of Western blot and other assays.更多还原