Recombinant single chain antibodies as an instrument to search proteins involved in the interaction of microsporidia and other intracellular parasites with an infected host cell

Abstract

Obligate parasitism of microsporidia close to fungi protists causing widespread diseases of animals and immunodeficient patients, significantly complicates studies of their relationships with an infected host cell. Since microsporidia cannot be cultivated outside the host cell, genetic manipulations with them are extremely difficult. At the same time, long adaptation of microsporidia to the intracellular lifestyle, drastic minimization of their metabolic machinery, acquisition of unique transporters to exploit a host cell make these parasites a very valuable object for such study and require a search for new methods of investigation. Here, we describe our experiment on the construction of the library of recombinant single chain antibodies (scFv fragments) against proteins of fat bodies of locusts Locusta migratoria infected by the microsporidia Paranosema (Antonospora) locustae. The representativeness of this library was about 108 E. coli transformants carrying different combinations of variable fragments of heavy and light chains of immunized mice immunoglobulins. The first results of the selection of scFv fragments from the constructed library by phage display technology demonstrated that this approach may be effective to search proteins involved in the interaction of microsporidia and other intracellular parasites with an infected host cell. Cloning of selected genes into the expression vector, transformation of E. coli and screening two hundred bacterial colonies revealed scFv fragments against several such candidate proteins to begin their study. Further experiments with the library should discover new variants of recombinant antibodies interacting with the parasite and host proteins.