[Cloning and expression of heavy- and light-chain variable region genes of monoclonal antibody specific for human fibrin].
OBJECTIVE
To obtain the minimum molecule having activity to bind human fibrin.


METHODS
The immunoglobulin heavy- and light-chain variable region (VH and Vkappa) genes were isolated from 8E5 hybridoma cells, which secreted a monoclonal antibody against human fibrin, by RT-PCR. An expression vector pOPE51-8E5 was constructed for the recombinant VH-Vkappa expression. The transformed E. coli JM 109 cells were propagated and induced by IPTG.


CONCLUSIONS
Expression product was found in the periplasmic space and inclusion bodies by SDS-PAGE and immunobloting. It was a 30000 single chain fragment (scFv) with antigen-binding specificity. The yield of the scFv was 28.9% of the total bacterial proteins.
Song
Z
Z
State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS and PUMC, Tianjin 300020.
Cai
Y
Y
Song
D
D
Xu
J
J
Wang
L
L
Yuan
H
H
Liu
J
J
Zhang
Y
Y
Lin
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D002460:Cell Line; D003001:Cloning, Molecular; D005337:Fibrin / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D005337:Fibrin