CLONING AND EXPRESSION OF A MURINE FAB FRAGMENT RECOGNIZING A DEFINEDLINEAR EPITOPE OF CHIRONOMUS-THUMMI THUMMI MAJOR ALLERGEN CHI-T-1-9

We have cloned and expressed in bacteria the genes coding for the Fab fragment of the monoclonal antibody (MoAb) M3. M3 is a murne IgG1 anti body reactive with the major allergen Chi t 1-9 of Chironomus thummi t hummi. The major allergen Chi t 1-9 is known to be an aggressive inhal ant allergen and causes type-I allergy. The immunoglobulin (Ig) fragme nt genes were cloned as a synthetic dicistronic operon. In this operon each gene is preceded by a bacterial signal sequence to direct the re combinant protein to the periplasmic space of the bacteria. The cloned genes were expressed in Escherichia coli using the strong T7-RNA-poly merase-based system. Sequence analysis revealed that the light and hea vy chains of MoAb M3 belong to the VchiII and VHIIC group of the Ig fa mily, respectively. Genes of the VchiII and VHIIC group are known to b e used in response to haptens. The apparent affinity constants of the parent antibody M3 and the recombinant Fab fragment are nearly equival ent.