[Gene cloning, expression and immunological identification of a single chain antibody fragment 3D3 against dengue...

Hybridoma 3D3 which secreted monoclonal antibody against Dengue type 3 virus was used to extract mRNA and then was reverse-transcripted into cDNA for amplifying heavy and light chain variable domain genes. The amplified light and heavy chain variable domain genes were connected by flexible linker to form a single chain antibody fragment (ScFv) gene of 750 bp, which was cloned into phage pCANTAB5 vector using the recombinant phage antibody system. The ScFv gene was expressed as fusion protein with phage g3p coat protein and displayed on the phage surface. The phagemid was used to transform competent E. coli TG1 cells and then infected with M13K07 helper phage to rescue the phagemid and antibody ScFv gene. 12 of the 20 randomized clones were shown to react with dengue type 3 virus by immunofluorescence.