Bacterial expression of a neutralizing mouse monoclonal antibody Fab fragment to a 150-kilodalton surface antigen of Entamoeba histolytica.
A mouse monoclonal antibody (MAb) (EH3015, IgG1 with a K light chain) prepared by hybridoma technology recognizes a 150-kD surface antigen of Entamoeba histolytica and inhibits adherence and cytotoxicity of the ameba to mammalian cells. The genes encoding the light chain and the Fd region of the heavy chain of the MAb were cloned and expressed in Escherichia coli. The plasmid used was designed for the expression of Fab with a hexa-histidine tag in the periplasmic space. Recombinant Fab fragments were purified and analyzed by an indirect immunofluorescence antibody test and Western immunoblot. The specificity of the recombinant Fab fragment was comparable with the parent whole IgG. In addition, the Fab fragments significantly inhibited the adherence of E. histolytica to erythrocytes. These results suggest that the production of a neutralizing MAb in Escherichia coli is practical and efficient with this expression system.
Tachibana
H
H
Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Takekoshi
M
M
Cheng
X J
XJ
Maeda
F
F
Aotsuka
S
S
Ihara
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000913:Antibodies, Protozoan / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002448:Cell Adhesion / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D015964:Gene Expression Regulation, Bacterial; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid