The creation of a recombinant single-chain antibody against α/β-hydrolase of microsporidium Paranosema locustae and immunolocalization of the enzyme in an infected host cell

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

“…PCR-amplification of DNA fragment encoding the last mentioned antibody and its analysis with restriction enzymes HaeIII, MspI, MseI, and HindIII (Fig. 5, A) showed similar digestion pattern with the gene of anti-hydrolase scFv fragment selected in the previous study (Dolgikh et al, 2017). However, comparative analysis of nucleotide sequences encoding two scFv fragments revealed six differences in amino acid compositions of the VH fragments.…”

“…It suggests that a portion of phages with antibodies specific to the host proteins was eliminated from the pool of selected viruses. PCR-amplification of selected scFv-encoding sequences using primers pSEX Nco for and pSEX Not rev showed that as in the case of the first library (Dolgikh et al, 2017) accumulation of viruses with abnormally small size antibodies took place during the selection process. In the case of plasmid DNA of the original library, amplification of about 800 bp fragments corresponding to the genes encoding full-length scFv fragments was observed (Fig.…”

“…5. Comparative analysis of sequences encoding (1) the new recombinant mini-antibody recognizing the proteins of about 63 kDa and 90 kDa from infected fat bodies of the locusts and (2) previously selected antihydrolase scFv fragment (Dolgikh et al, 2017). A -PCR-amplification of protein-encoding sequences followed by their incubation with four restriction enzymes showed similar digestion patterns; B -analysis of nucleotide sequences of two scFv fragments revealed some differences in their amino acid compositions (marked by the asterisks and enclosed in the squares).…”

“…To amplify sequences encoding VH and VL fragments of immunoglobulins we used frozen at -80 °C spleen of mice immunized for the creation of the first library (Dolgikh et al, 2017). The construction of the second library, selection of the phages, expression of scFv antibodies in E. coli and analysis of their antigen-binding activity were performed as described in the previous study (Dolgikh et al, 2017). To create a much more representative library, we scaled up about 200 times the amount of isolated total RNA, synthesized cDNA, amplified VH/VL genes, and prepared plasmid DNA.…”

“…To select the phages with specific antibodies, the proteins of fat bodies of infected locusts were immobilized on CNBr-activated Sepharose 4B (GE Healthcare Life Sciences, MA) instead of MaxiSorp polystyrene tubes (Nunc, Denmark) used in previous experiments (Dolgikh et al, 2017). To prepare antigen, fat bodies of infected locusts were gently disrupted in phosphate-buffered saline (PBS; 138 mM NaCl, 3 mM KCl, 1.5 mM KH 2 PO 4 , 8 mM Na2HPO4, pH 6.8) using a glass homogenizer and a freely matched Teflon pestle.…”