Genetically engineered single-chain Fvs of human immunoglobulin against hepatitis C virus nucleocapsid protein derived from universal phage display library.
Specific single-chain Fvs (scFvs) of human immunoglobulin that specifically recognized the recombinant hepatitis C virus (HCV) nucleocapsid protein were isolated from a large phage display antibody library. This universal library of genetically engineered filamentous phagemids displayed random pairings of the variable regions of both human heavy and light chain immunoglobulin in the scFv format. Specific clones were isolated by affinity selection with purified recombinant HCV protein fused to glutathione-S-transferase (GST). The GST-specific clones were excluded by blocking the phagemid library with GST prior to the selection. After 4 rounds of selection, the HCV-reactive clones were enriched by a factor of 100,000. About 4% and 9% of the clones from rounds 4 and 5, respectively, specifically reacted to the HCV portion of the fusion protein in an enzyme immunoassay. The specificity was confirmed by specific binding inhibition with plasma from an HCV-infected individual. Nucleotide sequence analysis of 3 HCV-specific clones indicated that all 3 clones contained an almost identical VH gene sequence which was derived from the VH3 germline gene family. These clones had different VL gene sequences of the lambda type. There were some differences between nucleotide and amino acid sequences of the HCV-specific scFv genes and those of the closest matched germline genes, indicating the presence of somatic mutation. This study illustrated the feasibility of using antibody engineering technology with the universal phage display library to isolate human antibodies with predefined specificity to important microbial pathogen which may be useful for future therapeutic purpose.
Songsivilai
S
S
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Dharakul
T
T
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005982:Glutathione Transferase / Q000235:genetics; D016174:Hepacivirus / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D018937:Hepatitis C Antibodies / Q000235:genetics / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment
D000939:Epitopes; D018937:Hepatitis C Antibodies; D007128:Immunoglobulin Fragments; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D005982:Glutathione Transferase