A recombinant human Fab expressed in Escherichia coli neutralizes rabies virus.
A recombinant human anti-rabies monoclonal antibody (MAb-57) Fab was prepared by cloning the heavy (Fd)- and light-chain domains into the same bacterial expression vector. To construct the recombinant Fab, mRNA was extracted from MAb-57-producing hybridoma cells, reverse transcribed, and then amplified by polymerase chain reaction (PCR) by using oligonucleotides specific for immunoglobulin heavy- and light-chain DNA sequences. PCR-amplified Fd-chain cDNA was fused, in frame, between a bacterial leader peptide (PelB) at the amino terminus and a 10-amino-acid peptide tag at the carboxy terminus. The PCR-amplified lambda-chain cDNA was also fused to the PelB leader peptide. The immunoglobulin Fab was then expressed as a dicistronic message in bacteria by using the isopropyl-beta-D-thiogalactopyranoside-inducible lactose promotor (lacZ). DNA sequencing was used to define the gamma-chain isotype (immunoglobulin G1) and VH (VHI) chain and VL (V lambda II) chain gene usage. The recombinant Fab (rFab57) specifically bound the rabies virus coat glycoprotein, while the Fd and lambda chains, when expressed individually, did not. The binding specificity of rFab57 was indistinguishable from that of the intact MAb in direct enzyme-linked immunosorbent assays; however, the dissociation constant of rFab57 for rabies virus protein G was approximately 1 log10 U lower than that of complete MAb-57 in competition enzyme-linked immunosorbent assays. A fluorescent-focus inhibition assay showed that bacterially expressed rFab was capable of neutralizing rabies virus strain CVS-11. We conclude that a human Fab expressed in bacteria maintains its specificity and biologic activity.
Cheung
S C
SC
Laboratory of Oral Medicine, National Institute of Dental Research, Bethesda, Maryland 20892.
Dietzschold
B
B
Koprowski
H
H
Notkins
A L
AL
Rando
R F
RF
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000914:Antibodies, Viral / Q000276:immunology*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011820:Rabies virus / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins