酵母菌Topoisomerase II單源抗體之製備

Abstract

摘要 本實驗中利用重組DNA的表現，得到相當量的混交蛋白質(hybrid protein)。其胺基端是Topoisomerase II，羧基端是β-galactosidase。利用Sephacry1-S-300膠質過濾管柱以及APTG-親和性管柱之純化，得到一極純之蛋白質，並經由聚丙烯醯胺膠電泳法SDS-PAGE分析其多勝鏈之分子量為160kd。將此純化後之蛋白質免疫鼴鼠BALB／c三次，並進行融合瘤細胞之製備，得到6個早抗原陽性反應之融合瘤細胞株。其中兩株經酵素連接免疫分析法分析為陽性反應較強者，乃進行單源抗體之大量製備。製備出的單源抗體，經純化後分析其亞型，再用酵素連接免疫沾染法(Western blot)證實此兩細胞株即2A3和1D5分泌之抗體乃特定於Topoisomerase II之單源抗體。此外，我們為了進一步瞭解，這兩株單源抗體和Topoisomerase II活性的關係。我們進行酵素活性分析實驗，結果顯示2A3和1D5對於Topoisomerase II活性有抑制的作用，此乃確信2A3和1D5確為Topoisomerase II之單源抗體細胞株。

ABSTRACT Sufficient quantites of a fusing protein TOP 2-β-Galactosidase has been prepared by expressing the recombinant DNA in E. coli. The recovery procedures include ammonium sulfate fractionation, molecular sieving (sephacryl S-300) chromatography and APTG-agarose affinity chromatography. The purified protein has a MW of 160 kd in SDS-PAGE. Monoclonal antibodies to the fusing polypeptide have been prepared. Two clones 2A3 (IgG1/k) and 1D5 (IgG2b/k) are specific to TOP 2. The activities of topoisomerase II from yeast are inhibited by both 2A3 and 1D5.