Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies

Abstract

Background Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin.ResultsIn this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2Δ1–25-His6) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2Δ1–25-His6 was obtained after purification by Ni2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2Δ1–25-His6. Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins.ConclusionsThe high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies

“…Instead, CaCo-2 cell damage was observed after the confluent monolayer of cultured cells came into contact with 10 μg/ml of the full-length PLC or a culture supernatant that was diluted up to four times. The data were in accordance with the results obtained for the CPB2 atypical recombinant protein, which was expressed in baculoviruses [24].…”

“…In addition, the best results were achieved in terms of production, satisfactory yield, and purity of the rBacCPA250-363H6 protein, when the cellular lysate was subjected to Ni-NTA His-select-affinity chromatography under denaturing conditions. As suggested for other recombinant proteins, such as CPB2 [24], this feature could be explained with respect to the folded protein by the significant exposure of the C-terminal 6xHis tag of the denatured antigen. As shown in Fig.…”

“…L21 (AACTCCTAAAAAAC CGCCACC) has both the vertebrate Kozak sequence (CCGCCACC) and the A-rich sequence (TAAAAAA) found in the polyhedrin leader sequence, and it is well known that the A-rich sequence increased the expression level in Sf9 cells [23]. In order to improve the translation efficiency of the baculovirus-based expression system, two different cis regulatory elements, a L21 consensus sequences and a selected Kozak sequence previously used in other studies [24] were inserted and the resultant levels of protein expression achieved were compared in this study. Monoclonal and polyclonal antibodies, that were obtained and directed against the purified non-toxic C-terminal rBacCPA250-363H6 recombinant protein, were tested to evaluate their reactivity and specificity in immunoblotting assays and their eventual use in home-made immunoenzymatic assays, which were able to detect native CPA from field strains of C. perfringens.…”

Probabilistic White Matter Atlases of Human Auditory, Basal Ganglia, Language, Precuneus, Sensorimotor, Visual and Visuospatial Networks

“…Direct comparisons between deterministic diffusion tractography and gold-standard tract-tracing methods in rhesus macaques have revealed that diffusion-based connectome reconstructions generally produce reasonable estimates of large white matter projections (Dauguet et al, 2007 ; van den Heuvel et al, 2015 ). However, because tensor-based deterministic tractography approaches yield sparse connectomes (high specificity with comparatively low sensitivity) and higher-order probabilistic tractography approaches yield dense connectomes (high sensitivity with comparatively low specificity), other groups have begun to study tradeoffs between connectome sensitivity and specificity (Zalesky et al, 2016 ). Initial studies in this regard suggest (both empirically and theoretically) that “specificity is at least twice as important as sensitivity when estimating key properties of brain networks, including topological measures of network clustering, network efficiency and network modularity” (Zalesky et al, 2016 ).…”

“…However, because tensor-based deterministic tractography approaches yield sparse connectomes (high specificity with comparatively low sensitivity) and higher-order probabilistic tractography approaches yield dense connectomes (high sensitivity with comparatively low specificity), other groups have begun to study tradeoffs between connectome sensitivity and specificity (Zalesky et al, 2016 ). Initial studies in this regard suggest (both empirically and theoretically) that “specificity is at least twice as important as sensitivity when estimating key properties of brain networks, including topological measures of network clustering, network efficiency and network modularity” (Zalesky et al, 2016 ). Therefore, although not perfect, the deterministic, tensor-based tractography approach used to generate our functionally-defined white matter atlases likely errs in the proper direction when it comes to connectome sensitivity vs. specificity tradeoffs.…”