Expression, purification and specific monoclonal antibodies preparation of diphtheria toxin A fragment.
- Author:
Jing OUYANG
1
;
Jian-Wei WANG
;
Chun-Xiao WANG
;
Li GUO
;
Hou-Zhen TUO
;
Ting CUI
;
Tao HONG
Author Information
1. Institute for Viral Disease Control and Prevention, Chinese Center of Disease Control and Prevention, Beijing 100052, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Bacterial;
genetics;
Antibodies, Monoclonal;
biosynthesis;
genetics;
Chromatography, Affinity;
Diphtheria Toxin;
immunology;
Escherichia coli;
genetics;
Female;
Immunotoxins;
isolation & purification;
Mice;
Mice, Inbred BALB C;
Peptide Fragments;
immunology;
Plasmids;
Recombinant Fusion Proteins;
biosynthesis;
isolation & purification
- From:
Chinese Journal of Biotechnology
2004;20(5):689-693
- CountryChina
- Language:Chinese
-
Abstract:
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.
