| 摘要 |
<p>The invention discloses generation of a single chain variable fragment (ScF v) antibody from a well-characterized monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, by cloning variable regions of the heavy (V H) and the light (V L) 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 colt strain TG-1 and expressed as a ~.about.30 KDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherich ia coli strain HB2151, where the same clone, designated A116, was expressed mainly a s 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 V L domain, upstream to the complementarity-determining 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 V L gene of A11 6, corresponding to framework-1 region. The mutagenized A116 was designated as MA116. The introduction of these three bases corrected a localized frame-shi ft 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 analyze d 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.</p> |
