Rational design, expression and biological activity assessment of a novel peptide based on ricin toxin antibody
- VernacularTitle:抗蓖麻毒素A链人源化单域抗体的设计、原核表达及生物活性检测
- Author:
Jianwei GUO
;
Jiannan FENG
;
Shuntao WANG
;
Cong MA
;
Zhengguang WANG
;
Xuebing JIANG
;
Beifen SHEN
- Publication Type:Journal Article
- Keywords:
Ricin toxin;
Single chain Fv;
Humanized single-domain antibody
- From:
Chinese Journal of Microbiology and Immunology
2008;28(7):650-655
- CountryChina
- Language:Chinese
-
Abstract:
Objective To design and express a novel peptide based on ricin toxin antibody in E. coli, and to evaluate its biological activity. Methods Based on the crystal structure of ricin toxin A chain (RTA) and the RTA-rRNA interact in the complex model, the steric conformation of RTA was theoretical modeled and its functional domain was preliminarily determined. The humanized single-domain RTA antibody was designed rationally by computer-guidod molecular design method. Its coding sequence was ob- tained by overlapping extension PCR, and cloned into the pET-32a vector. The fusion protein was then ex-pressed in E. coli BL21 (DE3), identified by Western blot, and purified with Ni-NTA agarose. The binding and neutralizing activity of this novel peptide for riein was evaluated by competitive ELlSA assay and MTT assay. Results A recombinant human single-domain antibody expressing a polypeptide against RTA in the CDR3 loop was designed. The fusion protein was successfully expressed in E. coll. The purified protein can bind to ricin, and neutralize its activity in SP2/0 viability assay. Conclusion The success of the novel pep-tide based on riein toxin antibody provides a novel method to develop new generation of ricin antagonists.
