PRODUCTION OF SINGLE CHAIN FRAGMENT VARIABLE (scFv) ANTIBODY AGAINST PARANOSEMA LOCUSTAE ALPHA/BETA-HYDROLASE AND IMMUNOLOCALIZATION OF MICROSPORIDIAN ENZYME IN THE INFECTED HOST CELL.

Abstract

Microsporidia is a widespread group of fungi-related intracellular parasites. Direct contact of the most microsporidia species with host cytoplasm suggests that these parasites may control physiological processes of infected cells by secretion of various proteins. In previous experiments, secretion of significant amounts of microsporidia Paranosema locustae alpha/beta-hydrolase into infected cells of Locusta migratoria fat bodies was demonstrated using polyclonal antibodies against the enzyme. However, heterologous expression of microsporidian hydrolase in yeast Pichia pastoris cells was not accompanied by its secretion. In this study, we have constructed library of recombinant single chain antibodies (scFv-fragments) against proteins of fat bodies of infected locusts and isolated mini-antibody specifically recognizing the studied enzyme using phage display technology. Immunoblotting and immunofluorescent microscopy with selected scFv-fragment confirmed secretion of two different in size forms of P. locustae alpha/beta-hydrolase into infected host cell. Prospects of scFv-fragment use to explore the role of microsporidian hydrolase in host-parasite relations and mechanism of its secretion are discussed in the paper.