title: Functional expression of Bacillus anthracis protective antigen in E. coli.
abstract: The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175-764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl beta-D-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.
authors.affiliation: Razi Vaccine & Serum Research Institute, Ahmadabad Street, P.O. Box 91735-174, Mashhad, Iran. f.vahedi@rvsri.ir
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Vahedi
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Sankian
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Mahmoudi
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000187:drug effects / Q000276:immunology; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin