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

Anna Serroni,Silvia Tofani,Giovanni Pezzotti,Antonio De Giuseppe,Mirella Luciani,Giulio Severi,Manuela Tittarelli,Chiara Francesca Magistrali,Martina Pellegrini,Luca Bano,Chiara Di Pancrazio

doi:10.1186/s12934-017-0707-8

Abstract

BackgroundClostridium 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.

Highlights

Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins
The high-throughput of purified atoxic recombinant C. perfringens beta-2 toxin (CPB2) produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies
We report for the first time, the expression of non-toxic atypical CPB2 toxins, lacking the putative signal sequence, in a baculovirus system and its use as an antigen for the production of polyclonal and monoclonal antibodies

Summary

Introduction

Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. 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. For this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium distributed ubiquitously in the environment and the gut of many healthy humans and animals. As observed in the expression of alpha, kappa and theta toxins in C. perfringens, the transcription of the cpb gene is regulated by the two-component VirR and VirS system and its secondary RNA regulator VRRNA [5, 10, 11]

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