白喉毒素A片段的表达纯化与单克隆抗体制备

白喉毒素 (Diphtheriatoxin ,DT)A片段 (DTA)是白喉毒素的酶活性区 ,也是DT类免疫毒素的关键结构域。DTA蛋白及其单克隆抗体在免疫毒素的毒性机理、检测与纯化研究等方面具有重要价值。通过在E .coli中表达了DTA ,经Q SepharoseFF和Ni²⁺ Sepharose两步层析纯化 ,得到纯度约为 90 %的融合蛋白。以DTA为抗原免疫BalB c小鼠 ,获得了分泌抗DTA特异单抗的杂交瘤细胞株 3B6和 3B9。单抗为IgG1亚型 ,滴度达 1∶10⁶ 以上 ,与DTA的结合可被抗DT马血清竞争抑制。抗DTA单抗用于免疫印迹试验 ,或制备成免疫亲和柱纯化基于DT的重组免疫毒素 ,均获得较好效果 ,为免疫毒素的研究奠定了良好基础

Expression,Purification and Specific Monoclonal Antibodies Preparation of Diphtheria Toxin A Fragment

Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.