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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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• Burkholderia pseudomallei causing Melioidosis and Shigella dysenteriae is the most common cause of diarrhea, and so far no effective vaccine has been produced against these two bacteria. The BLF1 protein of Burkholderia pseudomallei bacteria plays an important role in pathogenesis and infection. Shigella dysentery STxB is one of the most important factors in pathogenesis and also has an adjuvant role. Binding the BLF1 protein with STxB can be a good candidate vaccine. In this study, the antibody titers of BLF1-STxB and BLF1 proteins produced in rats were compared. 

 
Materials & Methods: In this experimental study, pET28a (+) - blf1-stxB and pET28a (+) - blf1 vectors were transformed into E. coli BL21 (DE3) and confirmed by PCR. The expression of blf1-stxB and blf1 genes was induced by IPTG and the proteins were injected into rats four times after purification of the protein by using an affinity chromatography column. Polyclonal antibodies produced in the serum of rats were measured.
 

• The produced recombinant proteins were approved by SDS-PAGE and Western blotting. ELISA results showed that antibody was produced against the BLF1 antigen and the antibody titer level increased by STxB binding to BLF1, compared with the antibody titer against the BLF1 antigen.

 
Discussion & Conclusions: The BLF1-STxB protein had higher antibody titer than BLF1. Due to the structural similarity of STxB subunit with its counterpart in E. coli, it can be a vaccine candidate against Burkholderia pseudomallei, Shigella dysenteriae, enterohemorrhagic E. coli, and enterotoxigenic E. coli.
 

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Introduction: Box jellyfish stings are painful and may be life-threatening. The venom of Chironex fleckeri contains a variety of bioactive proteins as well as two of the most abundant proteins, namely CfTX-1 and CfTX-2 which cannot be isolated easily using electrophoresis or chromatography techniques. Recombinant expression technology may offer an alternative to the isolation of native C.fleckeri venom protein. This study aimed at expressing C-CfTX1-STxB protein in Escherichia coli and assessing its antigenicity in Syrian mice.
 
Materials & Methods: Synthesis of the artificial CfTX1complete gene was prepared in plasmid pUC57. The C-cftx1 was cloned using a polymerase chain reaction (PCR) and subcloned with BamHI and SalI restriction enzyme sites in pET28a-stxB expression vector and transformed into E.coli. Gene expression was artificially induced by Isopropyl β- d-1-thiogalactopyranoside. After the purification of the protein and its injection into the Syrian mice, the amount of produced antibody was measured in the serum. The rats were also challenged by the venom of the jellyfish (i.e., Rhopilema nomadic).
 
Findings: In this experimental study, the C-CfTX1-STxB gene was cloned in the expression vector pET28a (+), sequenced by PCR, and analyzed by enzymatic analysis. Moreover, the produced recombinant protein was confirmed by Western blotting. The produced antibody in the serum was quantified using an enzyme-linked immunosorbent assay.
 
Discussion & Conclusions: After 60 days, the immunized mice tolerated 50x LD50 of jellyfish venom. Considering the ineffectiveness of cardiotoxicity and neurotoxicity of the recombinant protein, this produced protein can be suggested as a jellyfish venom vaccine candidate in Syrian mice or at a later stage of a clinical trial in humans.

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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.

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Introduction: The bacterium Burkholderia Pseudomallei is the cause of melioidosis disease. BLF1 plays an important role in the pathogenesis and infection of B. Pseudomallei. STxB has an adjuvant and carrier role and can be produced by mixing vaccine-candidate antigens with this adjuvant to produce a suitable vaccine. This study aimed to construct and evaluate the immunogenicity of trimethyl chitosan nanoparticles containing BLF1-stxB protein by subcutaneous injection.
Material & Methods: In this study, the expression of recombinant BLF1-stxB protein was induced in the expression host, and the protein was purified by affinity chromatography. Then, nanoparticles were fabricated by ion gelation method and the size and shape of nanoparticles were assessed by electron microscopy and injected subcutaneously into mice four times. Antibody titration was evaluated by indirect ELISA. BLF1 toxin was used for immunogenicity.
(Ethic code: 6272)
Findings: The results of this study showed that protein-containing nanoparticles have higher size and PDI, and lower zeta potential than protein-free nanoparticles. The protein charge in nanoparticles was about 65%. The highest antibody titer belonged to the group receiving protein without nanoparticles. The results showed a 75% conservation challenge of the nanoparticle-free protein group.
Discussion & Conclusion: This study showed that the nanoparticle form containing this recombinant protein leads to a weaker immune response, compared to the non-nanoparticle form by injection. The results of the challenge showed that this recombinant chimeric protein provides better protection when subcutaneously injected with an adjuvant.