Development and production of a recombinant mini-antibody against rabies virus

MANSILLA, F.C.; TURCO, C.S.; CAPOZZO, A.V.; HELGUERA, G.

Buenos Aires

Conferencia; XXIX International Conference Rabies in the Americas; 2018

Rabies is the zoonotic infectiousdisease with the highest case-fatality rate. Rabies diagnostics rely onlaboratory testing and make use of specific antibodies. Several rabies virus(RABV) neutralizing mouse-derived monoclonal antibodies (mAbs) have beenidentified for this purpose. However, mAb production in mammalian cell-culture has aconsiderable cost, and the use of mice to obtain mAbs from ascitic fluid isincreasingly restricted. In this scenario novel biotechnological strategies arepursued to improve the availability of these valuable diagnostic tools. We havedeveloped a specific recombinant mini-antibody against RABV formed by thevariable region of the heavy and light chains of a previously describedneutralizing mAb (CR57) reactive against epitope 1 of the viral glycoprotein G(RV-GP). Both light and heavy chains variable regionsof CR57 were covalently linked by a flexiblepeptide linker and fused to the maltose binding protein (MBP) to improve thesolubility of the polypeptide. Expression of MBP-scFv57 was optimized using anE.coli expression system. The recombinant mini-antibody was recovered from thesoluble fraction and purified using a maltose-affinity column. Yield was 75g/lof initial culture. The integrity and stability of the purified MBP-scFv57 wereverified by SDS-PAGE/western blot, confirming that MBP fusion also improved thestability of the recombinant mini-antibody. The purified mini-antibodyrecognized RV-GP in the membrane of transiently-transfected HEK-293 cells. Ourpreliminary results show that this mini-antibody is efficiently produced as asoluble and stable protein in bacteria and can be affinity-purified in one step,preserving its binding activity. This mini-antibody is meant to be used as a inexpensive reagent capable of detecting the presence of virus in tissue samplesor infected cells.