綠竹筍生長過程差異性蛋白質體及其抗體庫之建立

Abstract

融合瘤技術是常用的單株抗體生產技術，藉由B細胞與骨髓瘤細胞的融合，得到分泌專一性抗體的融合瘤細胞株，此技術於發明以來三十年沒有太大的改變。本實驗室於2006年提出抗體庫概念，並以綠竹筍水溶性蛋白質體免疫小鼠，由兩階段細胞融合得到192株單株抗體。本論文承襲抗體庫的概念，進一步提出差異性抗體庫的想法，希望製備出能夠區別綠竹筍生長過程中，各成長階段差異性蛋白質之對應抗體。第一階段實驗，以差異性吸附管柱製備差異性蛋白質之抗體庫，結果因為吸附後之蛋白質差異性不大而不甚理想。於是改變製備策略，第二階段實驗則改以流式細胞儀進行差異性分選。利用實驗室已建立的融合瘤細胞 (H7c、C7a、D5b)，先進行小規模預備實驗，以抗小鼠IgG抗體的FITC結合探針，確定融合瘤細胞會保留膜上抗體。然後以抗原標定的方式，成功將融合瘤細胞篩選出來。正式操作時，以綠竹筍之兩個生長階段的全體蛋白質免疫小鼠，分別進行細胞融合，於T80-flask培養3週，然後以流式細胞儀進行負篩選 (去除相同的抗體) 及正篩選 (挑出差異性抗體)，限數稀釋後再以免疫染色做確認。最後結果，得到數株單株抗體，但無法有效得到未出土或出土60公分綠竹筍專一性抗體，因此，此實驗技術及流程仍然做進一步改進。

Hybridoma technology is the most frequently used method for preparing monoclonal antibody. It is produced by fusing antibody secreting splenic B cells with myeloma cells. This technology was basically unchanged since its first introduction in 1975 by Kohler and Milstein. The concept of monoclonal antibody bank was proposed by our lab in 2006, and totally 192 specific monoclonal antibodies was collected by immunizing mice with the total water-soluble proteins from bamboo shoot, and then following an improved fusion and screening procedure. In this project, we further proposed the concept of differential antibody bank, in which the antibodies recognizing those proteins that were specific to variuos growing stages of bamboo shoots were screened out. First attempt to construct this bank by immunoaffinity column was not successful, since we cannot produce the protein banks with significant difference. Flow cytometer which could differentially sort out the cells labeled with specific proteins was utilized. In preliminary tests, we confirmed that hybridma cells could produce membrane-bound antibodies as detected by anti-mouse IgG-FITC antibody; and several established hybridoma lines (H7c, C7a, and D5b) could be sorted successfully by antigen labeling. Then we immunize mice with total proteins from bamboo shoots (BS0TP or BS60TP), and fuse the B cells with myeloma cells. After 3 wk cultured in T80-flask, the specific antibody producing lines were sorted out by negative selection and subsequently positive selection. The antibody produced was then checked by Western blotting after limiting dilution. Finally, we get some monoclonal antibody, but it can’t recognize BS0TP or BS60TP specifically. Therefore, we have to improve this technique and the procedure.