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


CONCLUSIONS
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.
Ge
Xiao-dong
XD
Department of Pathology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Liu
You-sheng
YS
Wang
Xiao-dong
XD
Wang
Chang-shong
CS
Deng
Jun
J
Li
Hong
H
D000209:Acute-Phase Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D007249:Inflammation / Q000188:drug therapy / Q000276:immunology; D008562:Membrane Glycoproteins / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D019151:Peptide Library; D000099231:Lipopolysaccharide-Binding Protein