Engineering and functional evaluation of a single-chain antibody against HIV-1 external glycoprotein gp120
- 4 June 2005
- journal article
- research article
- Published by Oxford University Press (OUP) in Clinical and Experimental Immunology
- Vol. 141 (1), 72-80
- https://doi.org/10.1111/j.1365-2249.2005.02826.x
Abstract
Summary: 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 (VH) and light (VL) chains were prepared by reverse transcription PCR and linked together with an oligonucleotide encoding a linker peptide (Gly4Ser)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 µg/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.This publication has 39 references indexed in Scilit:
- Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumorsBlood, 2002
- Effector Function Activities of a Panel of Mutants of a Broadly Neutralizing Antibody against Human Immunodeficiency Virus Type 1Journal of Virology, 2001
- Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domainNucleic Acids Research, 2001
- Isolation of Single-Chain Antibody Fragments Against Venezuelan Equine Encephalomyelitis Virus from Two Different Immune SourcesViral Immunology, 2001
- Characterization of Monoclonal Antibodies Against Carcinoembryonic Antigen (CEA) and Expression inE. coliHybridoma, 2001
- GP120: Biologic Aspects of Structural FeaturesAnnual Review of Immunology, 2001
- Enhancing antibodies in HIV infectionParasitology, 1997
- Splicing by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interestGene, 1997
- A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen bindingJournal of Immunological Methods, 1996
- Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding.The Journal of Experimental Medicine, 1991