Human single-chain Fv fragments from a combinatorial library using the loxP-Cre recombination system.
A human scFv display library has been constructed from peripheral blood lymphocytes of a patient suffering from Hashimoto's thyroiditis. Upon induction of Cre recombinase, the amplified VH and VL genes were recombined via two loxP sites inserted in amplification primers to construct in vitro scFv genes. Either soluble scFvs or scFvs displayed on phage were screened for binding to human thyroglobulin after two pannings with this antigen. Three scFvs were obtained which showed very similar nucleotidic sequences. The VH genes expressed display 96.4% nucleotide sequence homology with the germline VH251 gene, one of the two functional members of the small VH5 family and are mutated in sites already described as "selectively neutral" mutations and the VL genes are close to the germline DPL8 gene. These scFvs bind not only to human thyroglobulin but also to other self and exogenous antigens.
Biard-Piechaczyk
M
M
CNRS UMR 9921, UFR Sciences Pharmaceutiques et Biologiques, Montpellier, France. martinep@pharma.univ-montpl.fr
Teulon
I
I
Peraldi-Roux
S
S
Del Rio
M
M
Pau
B
B
Embleton
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005803:Genes, Immunoglobulin; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D013954:Thyroglobulin / Q000276:immunology; D013967:Thyroiditis, Autoimmune / Q000276:immunology*; D014764:Viral Proteins
D001323:Autoantibodies; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D014764:Viral Proteins; C055966:immunoglobulin Fv; D013954:Thyroglobulin; C045073:Cre recombinase; D019426:Integrases