Construction, expression and identification of the anti-idiotypic single chain variable fragment against Edwardsiella tarda
- VernacularTitle:迟缓爱德华菌抗独特型单链抗体基因的构建、表达及鉴定
- Author:
Hong QIN
;
Xiaohang JIN
;
Weiquan HUANG
;
Yulin LIU
- Publication Type:Journal Article
- Keywords:
Edwardsiella tarda;
anti-idiotype;
single chain antibody
- From:
Journal of Xi'an Jiaotong University(Medical Sciences)
2009;30(6):689-693
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct, express and identify the anti-idiotypic antibody single chain variable fragment (scFv) against Edwardsiella tarda. Methods By using RT-PCR method, the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody (mAb) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly_4ser)_3 linker, and the scFv in the orientation of V_L-linker-V_H was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia coli BL21(DE3), and confirmed by SDS-PAGE, Western blot and ELISA. Results The recombinant scFv could be expressed in E.coli BL21 (DE3) in a fusion protein pattern. The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded. The SDS-PAGE and Western blot analysis showed that the molecular had the binding activity to the antigen. Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21 (DE3), providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.
