As. Pac. J. Mol. Biol. & Biotech., Dec 1998 Vol. 6(2) : 79-88

Construction of a single chain variable fragment (ScFv) antibody recognizing the dengue 2 virus envelope protein

Hui Yee Chee and Sazaly AbuBakar*

Department of Medical Microbiology, Universiti Malaya Medical Center Kuala Lumpur, Malaysia

(Received 26 February, 1998 / Accepted 20 June 1998)

Abstract

A recombinant single chain variable fragment (ScFv) of the 3H5-1 monoclonal antibody recognizing dengue 2 virus envelope (E) protein was constructed using a bacteriophage expression system. The variable heavy (V_H) and variable light (V_L) chain genes were amplified and subcloned into the pGEM-T PCR cloning vector. The nucleotide sequences were then determined. Based on the sequencing results, new sets of primers for amplification of the VH and VL genes were designed incorporating the linker peptide, Not 1, Sfi 1, BamH I restriction endonuclease sites and the Flag peptide sequences. A second RT-PCR was performed using the newly designed primers and the resulting V_H and V_L DNA fragments were cloned into a phagemid, pCANTAB 5E. Bacteriophages expressing the ScFv which recognized dengue 2 virus envelope protein were rescued following 'panning'. Two recombinant phage clones which showed significant binding were obtained and soluble forms of the 32 kDa ScFv were expressed following infection of the HB 2151 Escherichia coli.

Key Words: 3H5, dengue, envelope, phage, Scfv