[Expression and neutralizing activity analysis of humanized single-chain antibody against anthrax protective antigen].
OBJECTIVE
In order to reduce the human anti-mouse antibody (HAMA) response, anti-anthrax protective antigen scFv-5E1 has been humanized.


METHODS
The authors have constructed a "humanized" antibody fragments by grafting the complementarity-determining regions (CDRs) of the murine anti-PA scFv-5E1 to human framework regions which showed maximal homology to the scFv-5E1 sequence. The humanized VH-5E1 and VL-5E1 DNA fragments were then joined by fusion PCR. The expression vectors named pET-hscFv-5E1 was constructed by cloning the humanized 5E1 scFv gene into the Nde I/EcoR I site. The transformed E.coli BL21(DE3) cells were propagated and induced by IPTG.


RESULTS
Expression product was found as inclusion body with expected size by SDS-PAGE analysis. Soulable scFv showed binding ability to the protective antigen by ELISA analysis. The purified scFv showed good neutralizing activity in a cell model.


CONCLUSIONS
The humanized scFv exhibited good binding and neutralizing activity. It lays a foundation for the development of therapeutic whole molecular antibody.
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
Bing
B
Li
Jun
J
Zhang
Jun-jie
JJ
Xu
Shu-ling
SL
Liu
Ling
L
Fu
Jian-min
JM
Li
Wei
W
Chen
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D022801:Complementarity Determining Regions; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D022801:Complementarity Determining Regions; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C030325:anthrax toxin