[Construction of human phage antibody library and screening and characterization of phage antibodies against N terminal fragment of human lipopolysaccharide binding protein].

AIM

To construct human phage antibody library and to prepare phage antibodies against N terminal fragment of human lipopolysaccharide binding protein (NH-LBP).

METHODS

NH-LBP was expressed in insect cells sf21 cultured with serum-free culture medium (SF-900 II) by BAC-TO-BAC baculovirus expression system. Human antibody library (Fab) was constructed by phage display system (pComb3H/VCSM13) and screened with NH-LBP.

RESULTS

NH-LBP was successfully expressed in insect cells sf21 cultured with serum-free culture and effectively purified via affinity chromatography column with TALON resin. About 8 mg NH-LBP was obtained. Human combinatorial phage display antibody library consisting of 5.0 x 10(8) CFU was constructed. After eight rounds of panning with NH-LBP we got 3 phage antibodies with high affinity for NH-LBP (DNA sequences and amino acid sequences of kappa chains of these antibodies were deposited in GenBank (AY337713 and AY337714).

CONCLUSION

Phage antibodies against NH-LBP were obtained successfully, which lays the foundation for preparing disulfide stabilized Fv fragments antibody (dsFv) against NH-LBP, understanding the function of LBP in vivo, and prevention and therapy of excessive inflammatory reaction.