Chang AH, Leslie K, Hoxie J, Cassol S; International Conference on AIDS.

Int Conf AIDS. 1996 Jul 7-12; 11: 60 (abstract no. Mo.A.1034).

B.C. Center for Excellence in HIV/AIDS, Vancouver, BC, Canada. Fax: 604-631-5646. E-mail: achang@hivnet.ubc.ca.

Objective: To determine the sequences of the antibody variable regions of three hybridoma cell lines, all of which produce monoclonal antibodies against the C' terminus of Nef protein, and to construct anti-Nef single-chain antibodies (SFV) in order to study the intracellular role of Nef in HIV-1 infection. Methods: The polymerase chain reaction (PCR) has been adopted to directly clone the VL and VH-specific moieties from hybridoma cell lines. These sequences were then inserted into a plasmid vector and sequenced using ABI's automated sequencer. The sequences were analyzed using Blast similarity search against the combined Non-redundant Nucleotide Database at National Center for Biotechnology Information (NCBI) Blast network server. The nucleic acid sequences of the variable regions (including both VL and VH) of hybridoma cell lines were also compared to each other by Fasta analysis. SFV were then constructed by connecting VL with VH, using a 15 amino-acid peptide linker (GGGGS)3. The SFV were expressed in pET-19b (Novagen) E. coli expression system, and the immunological activities were tested against the Nef protein. Results: The sequences from the variable regions, VL and VH of the three hybridoma cell lines, are unrelated to the sequences found in NCBI's database. There was 97.8% similarity (among 774 overlapping nucleotides) between AG11 and AE6, which were raised in the same fusion. But only 76.1% similarity (among 661 overlapping nucleotides) was found between AG11 and EH1. All of the three SFV expressed by E. coli could recognize recombinant Nef protein by ELISA assay. Conclusion: The recombinant SFV retained the immunological activities of their corresponding parent monoclonal antibodies. The similarity of the 3-dimensional conformation of the antibody variable regions is believed to be contributing to the EH1 monoclonal antibody recognizing an overlapping epitope with AG11 and AE6, rather than their linear amino-acid sequence similarities. By intracellularly expressing anti-Nef SFV in CD4 and monocytic cell lines, the SFV can be used to study the intracellular role of Nef in HIV-1 infection and to set up a gene therapy model for the potential treatment of AIDS.

Publication Types:

• Meeting Abstracts

Keywords:

• Amino Acid Sequence
• Antibodies, Monoclonal
• Base Sequence
• Cell Line
• Cloning, Organism
• DNA Primers
• Enzyme-Linked Immunosorbent Assay
• Epitopes
• Gene Products, nef
• Hybridomas
• Immunoglobulin Variable Region
• Polymerase Chain Reaction
• Recombinant Proteins
• genetics
• immunology

Other ID:

• 96921105

UI: 102217004

From Meeting Abstracts