Mohammadreza Rahmani; Shahram Nazarian; Hossein Samiei-Abianeh; Seyed Mojtaba Aghaie; Davoud Sadeghi
Abstract
Background: Stonefish (Synanceia spp.) are among the most venomous marine organisms. Their venom contains stonustoxin (SNTX), a heterodimeric toxin that induces severe hemolytic and myotoxic effects primarily mediated by its β-subunit.Objective: To produce a recombinant SNTX β-subunit ...
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Background: Stonefish (Synanceia spp.) are among the most venomous marine organisms. Their venom contains stonustoxin (SNTX), a heterodimeric toxin that induces severe hemolytic and myotoxic effects primarily mediated by its β-subunit.Objective: To produce a recombinant SNTX β-subunit and develop neutralizing polyclonal antibodies against SNTX.Methods: A DNA cassette encoding immunogenic regions of the β-sntx was designed using bioinformatics analysis, and codon-optimized for expression in E. coli. The construct was cloned into pET17b vector, and expressed in E. coli BL21 (DE3). The recombinant protein was purified via Ni-NTA affinity chromatography. For antibody production, rabbits were immunized subcutaneously with the recombinant protein emulsified in Freund's complete adjuvant, followed by booster doses at 2-week intervals. Antiserum was purified using protein G chromatography, and antibody titers were assessed by indirect Enzyme-Linked Immunosorbent Assay (ELISA).Results: Epitope mapping revealed key immunogenic regions within residues 124–654 of the β-SNTX subunit. Following codon optimization, the codon adaptation index (CAI) increased to 0.93. Recombinant protein production was confirmed by SDS-PAGE and Western blot demonstrating successful purification of a 73 kDa recombinant protein (including TRX/His-tags), with a yield of 40 mg/L. Immunization of rabbits elicited a strong polyclonal IgG response, with antibody titers reaching 1:25,600 following the third booster. Purified IgG (1.8 mg/mL) exhibited high sensitivity in ELISA, detecting the recombinant β-SNTX at concentrations as low as 31.25 ng.Conclusion: The recombinant β-SNTX subunit demonstrated strong immunogenicity, inducing high-affinity antibodies with specific binding activity against the native toxin. The resulting antiserum demonstrated significant neutralization potential, highlighting its promise for antivenom development.
Mona Khoshbakht; Mohammad Mahdi Forghanifard; Hossein Aghamollaei; Jafar Amani
Abstract
Background: Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy.Objective: This study aimed to engineer a recombinant chimeric fusion protein by combining ...
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Background: Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy.Objective: This study aimed to engineer a recombinant chimeric fusion protein by combining a nanobody-targeting domain with an exotoxin effector domain. The chimeric protein was designed to bind surface-expressed GRP78 on cancer cells, facilitating internalization and inducing apoptosis to inhibit proliferation and survival.Methods: Using a flexible linker, we designed two constructs linking VHH nanobody domains to Pseudomonas exotoxin (PE) domains II, III, and Ib. These constructs were then optimized for expression in E. coli BL21 (DE3) using the pET28a vector. Following the expression of the recombinant proteins, we purified them and tested their binding capability, cytotoxicity, and ability to induce apoptosis in breast cancer cell lines MDA-MB-231 and MCF-7, as well as in control cell lines HEK-293 and MDA-MB-468. The binding affinity was measured using a cell-based ELISA, internalization was assessed through Western blotting, cytotoxicity was evaluated by an MTT assay, and apoptosis was determined using flow cytometry with an Annexin V kit.Results: The immunotoxin specifically bound to cancer cells expressing csGRP78. The results of the cytotoxicity test showed that the cytotoxic effect of two constructs, I and II, depended on concentration and time. With an increase in both components, the effect of recombinant proteins also increased. Both constructs were able to penetrate and induce apoptosis in csGRP78+ cells.Conclusion: These immunotoxin structures showed therapeutic potential against GRP78-expressing cancers, making them suitable candidates for targeted therapy pending in vivo studies.
Mohammad Hadi Fakoor; Parviz Owlia; Seyed Latif Mousavi gargari; Azar Sabokbar
Abstract
Background: Pseudomonas aeruginosa is considered as the most severe cause of infections in burn patients and pneumonia infections. Objective: To study the protective effects of recombinant protein vaccine harboring the PcrV of P. aeruginosa in the mouse model of burn and respiratory infections. Methods: ...
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Background: Pseudomonas aeruginosa is considered as the most severe cause of infections in burn patients and pneumonia infections. Objective: To study the protective effects of recombinant protein vaccine harboring the PcrV of P. aeruginosa in the mouse model of burn and respiratory infections. Methods: Recombinant protein vaccine harboring the PcrV was expressed in the E. coli BL-21 strain. Mice were immunized with the purified recombinant protein, and the antibody titer was measured in the sera obtained from the immunized mice. Immunized and control mice werechallenged by active and passive immunization. The microbial counts in the lung, skin, liver, spleen, and kidney were compared with the control mice. Results: Bioinformatics analysis indicated that the PcrV protein was conserved in 1552 clinical and environmental isolates. Also, the isoelectric point (pI), molecular weight, and Grand Average of Hydropathy (GRAVY) score were analyzed. Mice were injected with recombinant protein, and serum from immunized mice reacted strongly with recombinant antigen at a dilution of 1:64000. The survival rate of mice infected with 5xLD50 of the P. aeruginosa increased significantly up to 75% in the standard strains (PAO1 and PAK), and the number of bacteria, especially in the internal organs (kidney, spleen, and liver) significantly reduced compared to the mice immunized with placebo. Conclusions: Our results demonstrated that the PcrV protein could be an effective candidate vaccine for the generation of antibody response against P. aeruginosa infection.
Fateme Sadri-Ardalani; Moslem Ahmadi; Azam Hemmati; Shaghayegh Emami; Samira Farid; Mohammad Mehdi Amiri; Mahmood Jeddi-Tehrani; Mahdi Shabani; Fazel Shokri
Abstract
Background: In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 ...
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Background: In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 protein boosting. In the present study, we evaluated the immunogenicity of different HER2 extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c mice. Objective: To investigate and characterize antibody responses to human recombinant proteins of HER2 extracellular subdomains in immunized mice. Methods: Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently, purified recombinant proteins were intraperitoneally injected in BALB/c mice with Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA, immunoblotting and flow cytometry. Results: All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay. Conclusion: The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.
Bahram Kazemi; Negar Seyed; Mojgan Bandehpour; Zarrin Sharifnia; Parviz Pakzad
Abstract
Background: Although a simple and direct method does not exist for the detection of chlamydial infections, there are situations in which reliable serological tests, with sensi-tivity related to a specific antigen, could be helpful. Objective: The aim of this study was to clone the first 1100 bp of the ...
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Background: Although a simple and direct method does not exist for the detection of chlamydial infections, there are situations in which reliable serological tests, with sensi-tivity related to a specific antigen, could be helpful. Objective: The aim of this study was to clone the first 1100 bp of the C. trachomatis outer membrane protein 2 (omp2) gene in order to prepare a recombinant protein for use in an ELISA system designed to recognize the anti- C. trachomatis antibody in patient sera. Methods: The PCR product of the chlamydial omp2 gene was cloned in pBluescript and its first 1100 bp was sub-cloned in the pQE-30 expression vector and induced by IPTG. The recombinant protein was purified by affinity chromatography and its purity was confirmed by SDS-PAGE, gel diffusion and western blot analyses. The purified protein was coated onto a polysty-rene microplate and tested by ELISA using patient serum. Results: We have cloned, over-expressed and purified biologically functional recombinant truncated Omp2 from C. trachomatis for use, as a species-specific recognition antigen, in an ELISA system. In this study we determined a cut-off value of 0.345 for this ELISA system using 55 negative sera and measured six positive sera at dilutions of 1:20-1:2560. Conclusion: As a species-specific recognition antigen, the over-expressed and purified recombinant truncated Omp2 from C. trachomatis performed well in an ELISA system.
