Construction, expression and characterization of a mono-specific bivalent diabody derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum
- VernacularTitle:日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体的构建表达与初步鉴定
- Author:
Yi ZHU
;
Jin ZHU
;
Zhenqing FENG
- Publication Type:Journal Article
- Keywords:
Schistosoma japonicum;
Anti-anti-idiotypic monoclonal antibody;
Diabody;
Expression;
Dot-ELISA
- From:
Chinese Journal of Schistosomiasis Control
1989;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a mono-specific bivalent diabody (scFv dimer) gene derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum and to express and characterize the protein.MethodsThe mono-specific diabody gene (D) was constructed by SOE (splicing by overlap extension) and using Gly_4Ser as a linker to join the C-terminus of the V_H to the N-terminus of the V_L.D was linked with prokaryotic expression vector pBAD/g. The target protein expression in E.coli TOP10 was induced by arabinose. Then a purification procedure for the target protein was carried out. The antigen binding activity of expressed product was detected with Dot-ELISA. ResultsThe V_H-G_4S-V_L (D) gene was confirmed by sequencing. The pBAD/g-D recombinant were determined by digesting with endonucleases and expected bands were identified. There were less soluble target proteins in the supernantes and higher target proteins in the pellets as inclusion body when separating the D expression proteins. And the insoluble fraction was recovered as a soluble, correctly processed protein by solubilising with 8 mol/L Urea. The molecular weight of the target protein was about 27 kD. The binding activity of the target protein to anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum was verified by Dot-ELISA. ConclusionThe purified protein from the constructed recombinant pBAD/g-D could interact specifically with antigen NP30. So the constructed mono-specific diabody has the part characteristics of anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum.
