Cloning, expression, sequencing, and functional enhancement of monoclonal ScFv antibody against venzuelan equine encephalitis virus (VEE)

The invention discloses generation of a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, by cloning variable regions of the heavy (VH) and the light (VL) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. Mab clone 1A4A1 was successfully cloned as ScFv in Escherichia coli strain TG-1 and expressed as a ~30 KDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151, where the same clone, designated A116, was expressed mainly as soluble periplasmic protein. The 30 KDa A116 displayed weak binding specificity to VEE. Sequence analysis revealed a frame shift in the N-terminal region of the VL domain, upstream to the complementarity-determnining region 1, as the probable cause of reduced activity. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5 prime region of the VL gene of A116, corresponding to framework-1 region. The mutagenized A116 was designated as MA116. The introduction of these three bases corrected a localized frame-shift in framework-1 region to consensus framework-1 amino acid sequence. Five MA116 clones (MA116-4, MA116-6, MA116-14, MA116-15, and MA116-16) have been analyzed for their reactivity to VEE antigen, with MA116-15 showing comparable reactivity to the parental 1A4A-1 Mab in recognizing VEE antigen. Sequence data revealed that only MA116-15 had incorporated the three intended base insertions without accumulating any other mutational changes.