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

Autor: Forti K; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy. k.forti@izsum.it.; Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia, Via San Costanzo 4, 06126, Perugia, Italy. k.forti@izsum.it., Cagiola M; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy., Pellegrini M; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy., Anzalone L; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy., Di Paolo A; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy., Corneli S; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy., Severi G; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy., De Giuseppe A; Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche 'Togo Rosati', Via G. Salvemini 1, 06126, Perugia, Italy.
Jazyk: angličtina
Zdroj: BMC biotechnology [BMC Biotechnol] 2020 Jan 28; Vol. 20 (1), pp. 7. Date of Electronic Publication: 2020 Jan 28.
DOI: 10.1186/s12896-019-0597-4
Abstrakt: Background: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms.
Results: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC.
Conclusions: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje