利用噬菌体抗体库筛选U251细胞血清应答基因蛋白特异抗体

利用噬菌体表面展示抗体库对不同血清处理U251细胞吸附的抗体进行差异筛选，筛选获得血清饥饿细胞吸附的阳性噬菌体克隆96个和血清饥饿后恢复血清培养细胞吸附的阳性噬菌体克隆82个。细胞免疫组化检测发现应答反应差异较大的抗体2个，即血清饥饿培养细胞特异反应的抗体1个（11号抗体）和血清饥饿后恢复血清培养细胞特异反应的抗体1个（2号抗体），其中2号抗体在恢复血清培养细胞中的应答反应强于血清饥饿培养细胞，是一个血清应答基因蛋白特异抗体，且在血清饥饿后恢复血清培养不同时间的U251细胞中具有一定的特异性反应。该研究为寻找与细胞周期调控有关的因子奠定了基础，同时对肿瘤的诊断和治疗研究也有重要意义。

Screening Serum Response Special Antibodies of U251 Cell Line from Surface Display Phage Antibody Library

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong response in serum recovered cells. The antibody No.2 had the distinctive response to the serum recovered cells in different incubation time (15min, 30min, 1h, 2h, 4h, 8h, 12h and 48h) after serum starvation. The results showed that No.2 antibody would be useful to research the factors of cell cycle regulation and apply to tumor diagnosis.