Construction and expression of human disulfide-stabilized Fv fragment gene to Rabies Virus
- VernacularTitle:人源抗狂犬病毒二硫键稳定性Fv抗体片段基因的构建与表达
- Author:
Xiaoling ZHAO
;
Jun YIN
;
Weiqiang CHEN
- Publication Type:Journal Article
- Keywords:
rabies virus;
disulfide-stabilized Fv fragment;
gene expression
- From:
Medical Journal of Chinese People's Liberation Army
1983;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To realize the construction and expression of human dsFv genes to rabies virus. Methods The genes of dsFv VH and VL were amplified by PCR-based point mutagenesis strategy. Then the genes were cloned into plasmid pET-22b (+). E. coli BL21(DE3) was transformed with the recombinant expression plasmid and the protein was induced in Luris-Bertani medium by addition of IPTG. The inclusion body proteins of VH and VL were denaturalized by GuHCl, and then re-naturalized in refolding solution to form dsFv fragments. Afterwards, we evaluated the antigen-binding activity of the dsFv and its stability as compared with its originative scFv. Results The fragment genes of dsFv to rabies virus were constructed successfully by PCR-based point mutagenesis strategy. Sequence analysis proved that cysteines were introduced into the position 44 aa of VH and position 100 aa of VL. The dsFv VH and VL genes were expressed in PET22b(+)/BL21(DE3). The VH and VL protein folded into the active dsFv antibody fragments which showed specific binding capability to rabies virus. Conclusion We succeeded in achieving the stabilization of the human scFv to rabies virus and obtaining the active human dsFv antibody fragments. This established a solid basis for further study of the biological activaty and clinical application of dsFv to rabies virus.
