Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

Cloning of variable regions of heavy chain and light chain of mAb 2-4 and constant regions of heavy chain and light chain of feline immunoglobulin: RNA isolation from cells and cDNA preparation were performed employing the method of [18] . The variable region genes of the mAb 2-4 (V H and V L ) were amplified by PCR from cDNA of hybridoma mAb 2-4 mRNA. The constant region gene of feline immunoglobulin heavy chain (C H ) was amplified from cDNA of feline peripheral blood mononuclear cell mRNA. The constant region gene of feline immunoglobulin light chain (C L ) was artificially synthesized by Life Technologies (Carlsbad, CA, U.S.A.) based on a published nucleotide sequence (Genbank AF198257.1) and inserted into the pMA-T plasmid. The primer sequences used for PCR are shown in Table 1 . V H , V L , C H and C L were individually cloned in pCR-blunt II-TOPO vectors using the Zero Blunt TOPO PCR cloning kit (Life Technologies).