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 10 8 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.

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

“…Three years ago, we constructed an immune scFv library against fat body proteins of locust Locusta migratoria infected with the microsporidium Paranosema ( Antonospora ) locustae and selected scFv fragments specific to three secretory proteins of the parasite [ 32 ]. The following sequencing showed that two of them had the same VL domain of a human IgG (unpublished data).…”

“…The gel-purified PCR products were cloned into the pSEX81 phagemid vector (Progen Biotechnik, Heidelberg, Germany) at the Nco I/ Hind III (VH domains) and Mlu I/ Not I (VL domains) restriction sites to construct an immune scFv library with a representativeness of about 10 million E. coli transformants (XL1-Blue MRF’ strain). All stages of library constructing, storage, infection with the M13 KO7ΔpIII hyperphage (Progen Biotechnik, Heidelberg, Germany), and phage production have been described previously [ 32 ].…”

“…The re-cloning of selected scFv genes into the pOPE101 plasmid (Progen Biotechnik, Heidelberg, Germany), their expression in E. coli , and the analysis of the ability of selected scFv antibodies to recognize Nb AAC1-3, Nb AAC1, Nb AAC2, Nb AAC3 by dot blotting and immunoblotting were performed as described previously [ 23 , 32 ]. The isolation of plasmid DNA from transformants producing Nb AAC-specific scFv-fragments was followed by sequencing of their genes with pOPE101seq primers ( Table 5 ) and alignment using Clustal W of MegAlign from DNASTAR’s Lasergene sequence analysis software [ 45 ].…”