Maziar Habibi-Pirkoohi; Saeid Malekzadeh-Shafaroudi; Hasan Marashi; Saeid Zibaee; Afsaneh Mohkami; Saba NejatizaXeh
Abstract
An Agrobacterium-mediated transient gene expression assay was carried out in alfalfa (Medicago sativa) leaves for expression of a chimeric gene encoding a part of capsid protein of Foot and Mouth Disease virus called VP1. The plant leaves were transformed via agroinfiltration procedure. The presence ...
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An Agrobacterium-mediated transient gene expression assay was carried out in alfalfa (Medicago sativa) leaves for expression of a chimeric gene encoding a part of capsid protein of Foot and Mouth Disease virus called VP1. The plant leaves were transformed via agroinfiltration procedure. The presence of the foreign gene and its expression in transformed plants were evaluated by polymerase chain reaction (PCR), real time PCR, protein Dot blot and ELISA. Moreover, gene expression in the transformed leaves was quantified by ELISA method. The results obtained in this investigation indicated high level of gene expression in alfalfa leaves, showing that transient gene expression can be applied as an effective and time-saving procedure for the production of recombinant proteins. The procedures for transformation, detection of recombinant protein and its application for molecular experiments are described in the study.
Amir Ghaffar Shahriari; Abdolreza Bagheri; Mohammad Reza Bassami; Saeed Malekzadeh Shafaroudi; Ali Reza Afsharifar
Abstract
Newcastle is a significant avian disease continuing to cause considerable loss. Developments in genetic engineering have led to plant-based platforms for human and animal vaccine production. Recombinant vaccine production in hairy root systems have several advantages over stable expression in whole plants, ...
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Newcastle is a significant avian disease continuing to cause considerable loss. Developments in genetic engineering have led to plant-based platforms for human and animal vaccine production. Recombinant vaccine production in hairy root systems have several advantages over stable expression in whole plants, including high growth rates, ready genetic manipulations, high levels of recombinant protein production, and the potential for bioreactor culture. In an attempt to develop a recombinant vaccine in hairy roots, the sequences encoding fusion (F) and haemagglutinin-neuraminidase (HN) epitopes of Newcastle disease virus were cloned in pBI121 expression vector which was then transferred into leaf disks of tobacco (Nicotiana tabaccum) 'Turkish' cultivar by means of Agrobacterium rhizogenes. Hairy roots developed on MS medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Incorporation of the heterologous gene in the genome of hairy roots was confirmed by PCR. Expression analyses were performed by real-time PCR at transcription level and by dot-blot and ELISA assays at translation level, all confirming the expression of the heterologous gene and production of the recombinant protein.
Maziar Habibi-Pirkoohi; Afsaneh Mohkami
Abstract
Green plants have emerged as ideal platforms for production of recombinant vaccine during recent decades. Various antigens relating to a large number of animal and human diseases have been studied in different plant species for production of recombinant vaccines. Despite the unique advantages of plant ...
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Green plants have emerged as ideal platforms for production of recombinant vaccine during recent decades. Various antigens relating to a large number of animal and human diseases have been studied in different plant species for production of recombinant vaccines. Despite the unique advantages of plant systems as green factories for production of recombinant vaccines, there are some major hurdles that have prevented commercial production of plant-based vaccines. In this review, theoretical background and practical applications of plant system for production of various recombinant vaccines are discussed.
Farzaneh Khaloiee; Poneh Pourfarzam; Iraj Rasooli; Jafar Amani; Shahram Nazarian; Seyed Latif Mousavi
Abstract
Shigella and Escherichia belong to the Enterobacteriaceae family which are the cause for most of the diarrheal cases in the world. Shigella can cause bacterial dysenteries and shigellosis. One of the most effective proteins for pathogenesis is invasion plasmid antigen C (IpaC). Other bacteria like Enterotoxogenic ...
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Shigella and Escherichia belong to the Enterobacteriaceae family which are the cause for most of the diarrheal cases in the world. Shigella can cause bacterial dysenteries and shigellosis. One of the most effective proteins for pathogenesis is invasion plasmid antigen C (IpaC). Other bacteria like Enterotoxogenic (ETEC), Enterohemorrhagic (EHEC), and E.coli can also cause diarrhea and produce intestinal disorders. Colonization factor antigen I (CFA/I), a critical virulence protein for these infections, has two subunits i.e. CfaB and CfaE. EHEC Attachment of bacteria is the main step of infection with intimin playing the key role in this function. This study was designed to elicit protection against the majority of diarrheal pathogens via development of polyvalent vaccine against Shigella, ETEC and EHEC. In silico techniques are as best tools to design new vaccines. For this purpose the immunogenic epitopes of CfaB, IpaC and Intimin were identified through bioinformatic tools and were then selected as major antigens to construct a chimeric protein (CII). The humoral and cellular immunities were analyzed bioinformatically. Prediction of allergens and mapping of IgE epitopes were carried out. The bioinformatic analysis showed each domain was folded separately in fusion structure. CII had many T and B cell epitopes in both linear and three-dimensional structures. This prediction of the chimeric construct had the potential to induce CD4+ and CD8+ immune responses against these pathogens. In addition CII could be accessible to surveillance by the immune system in mouse and human. In conclusion, in silico analysis showed that this chimeric protein can be used as a vaccine against Shigella, ETEC and EHEC simultaneously.
