Heterologous expression of scFv fragment against Vairimorpha (Nosema) ceranae hexokinase in Sf9 cell culture inhibits microsporidia intracellular growth

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

“…To construct an immune library of scFv Abs specific to Nb AAC1-3 fragments, we purified this chimeric protein of any bacterial impurities and immunized several mice to select a variant with high content of IgGs against the outer loops of each of the three N. bombycis ATP/ADP transporters. The representativeness of the constructed library (10 7 transformants) was also doubled compared to the previously constructed one against the microsporidium Vairimorpha ceranae hexokinase [ 23 ]. Despite this, we failed to isolate any recombinant Abs specific to loops of the second and third N. bombycis transporters.…”

“…The following sequencing showed that two of them had the same VL domain of a human IgG (unpublished data). Recently, we also selected a recombinant Ab against the microsporidium V. ceranae hexokinase containing this VL domain from the commercial vector pSEX81 [ 23 ], and in this study we found it in two anti- Vc AAC1 scFv fragments. Some characteristics of such scFv Abs with an “artificial” VL part should be similar to those of single domain Abs (sdAbs) carrying natural VHs of camelids [ 33 , 34 ] and cartilaginous fishes [ 35 , 36 ], or to “camelized” nanobodies [ 37 , 38 ].…”

“…Aspirated eluates were immediately neutralized with the same volume of 1 M Tris-Cl (pH 7.5) and stored on ice until bacteria infecting. E. coli XL1-Blue MRF’ cells were infected with eluted phages as described previously [ 23 ]. Their re-infection with helper phage, PEG precipitation, incubation with immobilized antigen, elution of bound phages, followed by re-infection of bacteria allowed for the second and third rounds of selection.…”

“…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 ].…”

“…S. frugiperda (Lepidoptera: Noctuidae) derived Sf9 cells from the ECACC General collection (ECACC 89070101) were cultured in Sf-900III SFM (Thermo Fisher Scientific, Waltham, MA, USA) and regularly maintained according to the manufacturer’s instructions. Transformation (lipofection) of adhesion cell cultures with 10 µg of pure plasmid DNA and selection of blasticidin-resistant transformants were performed as described previously [ 23 ].…”