The Journal of Immunology, Vol 150, Issue 2 469-479, Copyright © 1993 by American Association of Immunologists

ARTICLES

Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecules

MM Brigido, M Polymenis and BD Stollar
Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111.

A plasmid vector was constructed for the expression of a single chain Fv domain of mouse mAb to Z-DNA (antibody Z22), which is encoded by VH10 and V kappa 10 gene family members along with Dsp2, JH4, and J kappa 4 segments. The vector coded for a PhoA secretion signal, VH segment, flexible peptide linker, VL segment, (His)5, and a protein A domain. Unique restriction sites allowed exchange of the segments as cassettes. Bacteria transformed with the vector secreted soluble recombinant Fv with specific Z-DNA-binding activity. When the L chain of Z22 was replaced with a library of splenic VL cDNA from a mouse immunized with Z-DNA, only a light chain closely resembling that of the original Z22 (differing at six amino acid positions) yielded Fv with Z- DNA-binding activity. The Fv with this L chain replacement had a lowered affinity, but remained selective for Z-DNA. Replacement of the Z22 H chain with a mixture of 11 VH10-encoded H chains yielded two Z- DNA binding clones, but they bound B-DNA and denatured DNA as well as Z- DNA. The replacement clones indicate the importance of the H chain CDR3 and particular VH-VL combinations in formation of specific antibodies to Z-DNA.

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