| Products/Services Used | Details | Operation |
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| Recombinant Proteins | IBsTNFα were produced as described previously (Torrealba et al., 2016). Briefly, E. colicarrying the TNFα expression plasmid was cultured in LB broth with ampicillin (100 μg/ml) at 37 °C. When OD550 reached 0.5, recombinant protein expression was induced with 1 mM isopropyl-D-thiogalactoside (IPTG, Panreac) for 3 h. To purify IBs, enzymatic and mechanical disruption steps, were carried out as detailed in (Torrealba et al., 2016). Finally, samples were centrifuged at 15,000 ×g for 15 min, and pellets containing purified IBs were stored at −80 °C. IBs were checked for contamination on LB plates without antibiotic. Protein was quantified by western blot using an anti-His-tag antibody (Genscript A00186-100) and a standard curve using a recombinant protein and Quantity One software (Bio-Rad). | Get A Quote |
Waterborne pathogens are a constant threat to fish. Fish mucosa are the point of entry for the majority of microbes and are key in mounting an effective immune response to block invasion, colonization and spread. In the search for tools to enhance immune responses we have evaluated nanostructured TNFα protein nanoparticles (IBsTNFα) as potential mucosal adjuvants. IBsTNFα are non-toxic, stable, protein-based biologically active nanomaterials which are cheap to produce in microbial cell factories. Here we test the in vivo uptake of IBsTNFα in zebrafish larvae by bath immersion and in adult zebrafish by oral gavage. We used flow cytometry, histology and confocal microscopy, to show IBsTNFα can be effici... More
