Engineered resistance to Nosema bombycis by in vitro expression of a single-chain antibody in Sf9-III cells

Abstract

Nosema bombycis is a destructive, obligate intracellular parasite of the Bombyx mori. In this study, a single-chain variable fragment (scFv) dependent technology is developed for the purpose of inhibiting parasite proliferation in insect cells. The scFv-G4, which we prepared from a mouse G4 monoclonal antibody, can target the N. bombycis spore wall protein 12 (NbSWP12). Indirect immunofluorescence assays showed that NbSWP12 located mainly on the outside of the N. bombycis cytoskeleton, although some of it co-localized with β-tubulin in the meront-stage of parasites. When meront division began, NbSWP12 became concentrated at both ends of each meront. Western blotting showed that scFv-G4 could express in Sf9-III cells and recognized native NbSWP12. The transgenic Sf9-III cell line showed better resistance than the controls when challenged with N. bombycis, indicating that NbSWP12 is a promising target in this parasite and this scFv dependent strategy could be a solution for construction of N. bombycis-resistant Bombyx mori.

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

“…Many of the constructed scFv fragments demonstrated an ability to suppress the development of malaria plasmodium (Isaacs et al, 2011, Sumitani et al, 2013, cryptosporidia (Pokorny et al, 2008), trypanosomes (Ayub et al, 2012) and other intracellular parasites. Recently, the first data on suppressing of the intracellular development of entomopathogenic microsporidia by means of scFv-antibodies were published (Huang et al, 2018). In this study, we showed that scFv fragments can be used also to investigate the molecular machinery exploited by the intracellular protists to control physiological processes and molecular programs of an infected host cell.…”

Peer Review #4 of "A secretory hexokinase plays an active role in the proliferation of Nosema bombycis (v0.1)"

“…Nbβ-tubulin was used as a marker protein to distinguish the meront stage from mature spores. Meronts can be labeled using the Nbβ-tubulin antibody, while the mature spores can be observed visibly in differentialinterference microscopy (DIC) (Chen et al 2017;Huang et al 2018). The IFA demonstrated the NbHK can be secreted into host cells (Fig 2).…”

“…Then, the Manuscript to be reviewed products were diluted ten times as templates. Quantitative PCR was amplified using Hexokinase- (Huang et al 2018). The standard curve covered four orders of magnitude for the starting quantity (1.3 × 10 3 -10 6 ).…”

“…primers, and reference gene primers SSU-qF 5'-CTGGGGATAGTATGATCGCAAGA-3'and SSU-qR 5'-CACAGCATCCATTGGAAACG-3'(Huang et al 2018). Quantitative PCR was performed with following program: a predenaturation of 95 °C for 2 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 20 s (LightCycle 96, Roche, Switzerland).…”

Vairimorpha (Nosema) ceranae (Opisthosporidia: Microsporidia) in vitro Infection of Sf9 Insect Cell Line as an Experimental Model of Parasite – Host Interrelations

“…While the search for new effective chemicals active against V. ceranae is being continued [3], alternative approaches to suppress microsporidia intracellular development have also been suggested. Those are based on RNA interference [4][5][6] and single-chain antibodies (scFvs) [7]. The latter technology was successfully applied to decrease the mosquito Anopheles stephensi infection levels using scFvs against two surface proteins of apicomplexan parasite Plasmodium falciparum [8].…”