I.K. Biriukova1,2*, A.B. Shevelev2, M.V. Zylkova2, V.N. Trifan3,
A.A. Lebedeva2, A.V. Belyakova4, E.E. Kulikov1 and A.V. Letarov1

1RC Biotechnology, Russian academy of Science, Moscow 119071, Russian Federation.
2Chumakov institute of poliomyelitis and viral encephalitides, Moscow 142782, Russian Federation.
3State enterprise Orel biofactory, Orel 302501, Russian Federation.
4Innotech-21 LLC, Moscow region 140000, Russian Federation.

Abstract

The protective antigen (PA) of B. anthracis is a subunit of a complex toxin that mediates recognition of the eukaryotic cell receptors. Once bound to the receptor, PA recruits effector subunits (lethal and edema factors) and ensures their transportation to the cytoplasm of a target cell. The PA-neutralizing antibodies provide protection against the anthrax infection. Therefore PA is the main antigen used in vaccines. The 83 kDa PA consists of four domains. Domains I and II carry the major epitopes recognized by the protective IgGs from immunized humans and animals. We developed an integrative recombinant construct WPag201-SW that contains a fragment of PA gene encoding domain I (PA20). This construct allows production of this polypeptide in B. subtilis. Expression of PA20 is controlled by the Bacillus-specific promoter of cell wall bound subitlisin WprA. The secretion is mediated by the natural PA leader peptide. Due to the optimal position of the truncation point, we achieved an efficient secretion of a soluble protease-resistant protein to the medium. This protein was shown to react with the immune sera risen against full-length PA. The B subtilis strain carrying WPag201-SW construct is suitable for the large-scale production of the PA domain I fragment that can be used in test systems for serological detection of anti-PA antibodies. The live spore culture, producing PA domain I peptide, can be considered as a candidate to attenuated veterinary vaccine. Such a strain is of an urgent necessity for anthrax prophylaxis in cattle.

Keywords: Anthrax, vaccine, protective antigen, Bacillus anthracis, Bacillus subtilis.