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.
Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or “camelization” of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.
Sunn pest or Sunn bug, Eurygaster integriceps Put., salivary gland proteases are responsible for the deterioration of wheat flour quality during dough mixing, resulting from gluten hydrolysis. These proteases are highly heterogeneous and show low sensitivity to most types of proteinaceous inhibitors, meaning that such inhibitors cannot be used to prevent gluten damage. The present study describes the generation of a specific peptide antibody, raised against the active center of the recombinant gluten‐hydrolyzing protease (GHP3). The recombinant protein, encoding two repeats of the GHP3 sequence element involved in forming the S4 pocket and binding of substrate at position P4, was designed and expressed in Escherichia coli. The antibodies raised to this recombinant protein showed inhibitory activity against the GHP3 protease. The results indicate that it is possible to design specific antibodies to inhibit wheat‐bug gluten‐hydrolyzing proteases.
RNA-interference, or suppression of gene expression by small RNAs, was originally described in Caenorhabditis elegans in 1998 and is currently widely considered for use in plant protection. The use of double-stranded RNA molecules as an inducer of the RNA interference pathway in insect pests potentially allows employing them as active ingredients in modern pesticides. Genetically modified crops expressing dsRNA have been developed as commercial products with a great potential in insect pest management. Alternatively, some nontransformative approaches, including foliar spray and chemigation, are also suitable for practical applications. This review explains the mechanism of artificially induced RNA interference and existing strategies for the delivery of small RNAs to target insects within the framework of plant protection.
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