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)
2003;0(06):-
- 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(Gly4Ser)3 linker,and the scFv in the orientation of VL-linker-VH 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 weight of scFv protein was 27 ku.Indirect ELISA confirmed that the scFv 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.
