title: Cloning, expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody.
abstract: Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni(+)-NTA affinity chromatography (95-98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.
authors.affiliation: Faculty of Basic Sciences, Biology Research Center, Imam Hossein University, Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Rezaee
authors.forename: Hossein
authors.initials: H
authors.lastname: Honari
authors.forename: Mohammad Reza Ashrafi
authors.initials: MR
authors.lastname: Kooshk
mesh_terms: D000818:Animals; D000917:Antibody Formation; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008297:Male; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins