Engineering and functional evaluation of a single-chain antibody against HIV-1 external glycoprotein gp120.
The HIV-1 envelope glycoprotein surface subunit gp120 is an attractive target for molecular intervention. This is because anti-HIV-1 gp120 neutralizing antibodies display the potential ability to inhibit HIV-1 infection. The present investigation describes the construction of a genetically engineered single chain antibody (scFv102) against HIV-1 gp120, its expression and functional evaluation. The parental hybridoma cell line (102) produces an immunoglobulin directed against the conserved CD4-binding region of gp120. cDNAs encoding the variable regions of the heavy (V(H)) and light (V(L)) chains were prepared by reverse transcription PCR and linked together with an oligonucleotide encoding a linker peptide (Gly(4)Ser)(3) to produce a single chain antibody gene. The resulting DNA construct was cloned into a prokaryotic expression vector (pET28) and recombinant scFv102 was expressed in Eserichia coli as an insoluble protein. The denatured scFv102 was refolded and purified by immobilized metal ion affinity chromatography. Purified scFv102 had the same specificity as the intact IgG in immuno-blotting assays and immuno-fluorescence (IF) detection, but ELISA analyses demonstrated the affinity of scFv102 to be 5-fold lower than that of the parental monoclonal antibody. In neutralization assays, scFv102 at concentrations lower than 40 microg/ml exhibited efficient interference with viral replication and inhibition of viral infection (90%) across a range of primary isolates of subtype B HIV-1. These results suggest that the constructed anti-HIV-1 gp120 scFv102 has good biological activity and can potentially be used for in vitro diagnostic and in vivo therapeutic applications.
Wang
H W
HW
Department of Immunology, Southern Medical University, Guangzhou, 510515, PR China.
Cole
D
D
Jiang
W Z
WZ
Jin
H T
HT
Fu
N
N
Chen
Z L
ZL
Jin
N Y
NY
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D004306:Dose-Response Relationship, Immunologic; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology* / Q000502:physiology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000276:immunology
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins