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Introduction: Anthrax is a zoonotic disease. Bacterium Bacillus anthracis is the causative agent of the fatal disease. At present, the protective antigen (PA) is used as an effective vaccine against anthrax. Domain 4 of this antigen together with domain 1 of lethal factor (LF) are the potent immunogens of this bacteria and are as suitable candidates of vaccine against it. Our aim in this study is the cloning of protective antigen domain 4 (PAD4) genes and fusion of it with lethal factor domain 1 (LFD1) gene of the bacteria to evaluate their capability in protective immunity induction.

Materials & methods: In this experimental study, we used a recombinant pGEM-T easy vector containing LFD1 gene. Then PAD4 gene was amplified and isolated by PCR and cloned into another pGEM-T easy vector, separately. After that, ligation of PAD4 gene and LFD1 gene was done in mentioned vector with determination of LFD1 orientation and by PstI/XbaI restriction sites. The recombinant construct, resulted from these genes was sub-cloned into pET28a expression vector using BamHI/ XhoI restriction enzymes and after determination of genes orientation, the expression host BL21 was transformed by this recombinant vector.

Findings: First cloning and fusion of PAD4 and LFD1 gene fragments were successfully carried out in pGEM-T easy vector and after the confirmation of mentioned process by both of enzymatic digestion and PCR methods, the result recombinant construct was sub-cloned into pET28a.

Discussion & Conclusions: Since PAD4 and LFD1 are immunogenic regions, expression of the recombinant construct resulted from these ligated genes can be proposed as proper candidate of anthrax vaccine for induction of protective immunity.

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Introduction: Anthrax is a zoonotic disease and vaccine production is one way to protect people against this bacterium. This study investigates the application of copolymer nanocapsule (PEG-PLA) systems with controlled release specificity for a recombinant protective antigen and lethal factor of Bacillus anthracis to provide a vaccine candidate.
Material & Methods: In this experimental study, the dual solvent evaporation emulsion method was used to produce nanocapsules. Moreover, zeta potential and size of nanoparticles, loading efficiency of nanoparticles, recombinant protein release pattern, the probable effect of PLA-PEG nanoparticle production on the shelf life of recombinant proteins were investigated in this study. Mice were used as test and control samples for antibody production and immune response evaluation.
(Ethic code: 9727250)
Findings: The results of this study showed that mixed proteins (LFD1+PAD4) loaded on PEG-PLA block copolymer had an average size of 109 nm, the zeta potential of -27.7 mV, and PDI=0.394. In this study, it was shown that the release of these antigens was carried out in two stages of rapid and slow release. The release of the proteins was estimated at about 20% on the first day and 78% on the 49th day. The titers of antibodies produced in the serum of the mouse groups against these antigens at defined intervals were significantly different from each other.
Discussion & Conclusion: The results of this study suggest the application of PEG-PLA block copolymer nanocapsules containing mixed recombinant proteins of protective antigen and lethal factor of Bacillus anthracis. Due to less degradation and more protection of antigen activity in nanoparticles, compared to traditional methods, as well as fewer repeat injections, higher specificity, reduced side effects, lower cost, and slower release rate, the use of nanoparticles is a good option to replace traditional methods.