Cloning, expression, sequencing, and functional enhancement of monoclonal ScFv antibody against Venezuelan equine encephalitis virus (VEE)
US6818748B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Mar 13, 2002 |
| Grant date | Nov 16, 2004 |
| Priority date | — |
| Expiry date | Mar 13, 2022 |
Classification
- Technology area (CPC Y)Emerging Cross-Sectional Technologies
- CPC primaryY10S530/866
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
A single chain variable fragment (ScFv) antibody from a monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, is generated by cloning linked variable regions of the heavy (VH) and the light (VL) chain antibody genes. Mab clone 1A4A1 in E. coli strain TG-1 was successfully cloned as ScFv. Results were reproduced in E. coli strain HB2151, expressing the same clone, A116, though displaying weak binding specificity to VEE due to a frame shift in the N-terminal region of the VL domain, upstream to the complementarity-determining region 1. 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, producing mutant MA116, correcting a localized frame-shift in framework-1 region to consensus framework-1 amino acid sequence. A MA116 clone, MA116-15, shows comparable reactivity to the parental Mab in recognizing VEE antigen.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.