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
Volume 14, Issue 2 , June 2017, , Pages 99-110
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
Volume 5, Issue 3 , September 2008, , Pages 148-155
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.
