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PURPOSE: Newcastle disease (ND) represents a major viral disease across the world which imposes high costs to poultry producers for vaccination. Hemagglutinin-neuraminidase (HN) and fusion (F) proteins are the major immunogenic epitopes of Newcastle disease virus and hence, have been the main targets for development of anti-ND vaccines. This paper reports transient expression of a synthetic gene composing of four tandem repeats of HN and three tandem repeats of F epitopes in maize leaves as initial step toward production of recombinant vaccine against ND. MATERIALS AND METHODS: The synthetic gene was cloned in pBI121 plasmid to yield an expression vector. The vector was sophisticated by the addition of AUG codon, polyhistidine-tag, tobacco mosaic virus omega sequence, stop codon, and restriction sites. Leaf transformation was conducted by the agroinfiltration method. Molecular detection assays including polymerase chain reaction, reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were carried out to evaluate transgene expression in infiltrated leaves of the corn plant. RESULTS: The result obtained in this research revealed that the transgene was transcribed and translated in maize leaves only 48 hours after infiltration. In the second phase of the experiment, the expressed protein was injected into rabbits. The result of the ELISA assay indicated induction of immune response in the rabbits after injection with the heterologous protein. CONCLUSION: These results confirm the feasibility of agroinfiltration for transient gene expression of viral epitopes in monocot plants which naturally resist stable transformation by Agrobacterium tumefaciens. Practical implications of this finding are discussed in detail and some recommendations for future studies are proposed.
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BACKGROUND: Abiotic environmental stresses, especially drought stress, is one of the most important problems in arid and semi-arid regions. Like other major crops, Brassica napus is vulnerable to drought stress. OBJECTIVE: The present study was conducted to evaluate efficacy of Sargassum angustifolium extract on mitigating adverse effects of drought stress on B. napus seedlings during vegetative growth under greenhouse conditions. MATERIALS AND METHODS: Seedlings were periodically sprayed with the seaweed extract until they reached 7-leaf stage. Then water deficit stress was imposed and measurements were performed at morphological, biochemical and molecular levels on three phases: 80% field capacity for 20 days (Phase I), 60% field capacity for 20 days (Phase II) and 40% field capacity for 20 days (Phase III). Real-Time PCR assay was carried out to monitor the changes in expression of the genes involved in proline biosynthesis. RESULTS: Morphological measurements revealed that seaweed treatment improved shoot height and dry weight compared to control (p<0.05). Biochemical analyses indicated that foliar application of seaweed extract significantly enhanced the photosynthetic pigments' content, free radical scavenging and superoxide dismutase activity (p<0.05). Moreover, proline content was significantly increased in plant tissues treated with seaweed extract (p<0.05). The results of Real-Time PCR assay showed that the increase in proline content is due to enhanced expression of P5CS which is involved in biosynthesis of proline, and to decreased expression of PRODH which catalyzes proline degradation. CONCLUSIONS: Overall, the results obtained in this research suggest that application of S. angustifolium extract as a biostimulant is able to protect canola seedlings against deteriorating effects of drought stress.
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BACKGROUND: Newcastle disease is a major avian disease that causes enormous economic loss in poultry industry. There have been a number of reports on the suitability of plant-based recombinant vaccine against this disease. Fusion (F) and hemagglutinin-neuraminidase (HN) epitopes of the Newcastle disease virus (NDV) represent the major immunogenic sites for development of recombinant anti-ND vaccines in plant hosts. OBJECTIVES: The main objective of this research was to evaluate the ability of a recombinant anti-ND vaccine in induction of immune responses in animal model. MATERIALS AND METHODS: In this study, immunogenicity of recombinant fusion (F) and hemagglutinin-neuraminidase (HN) epitopes of the Newcastle disease virus (NDV) is investigated in an animal model. The corresponding genes encoding amino acids 65-81 of the F protein and 346-353 amino acids of HN were expressed in tobacco seedling using agrobacterium-mediated transformation. Expression of the foreign gene in the tobacco seedlings was investigated by a number of molecular assays including Real-Time PCR and ELISA. Transgenic plant extract was used to induce immunogenic response in animal model. RESULTS: Integration of the foreign gene in plant host genome was confirmed by polymerase chain reaction (PCR). Expression of the foreign recombinant protein was confirmed by Real-Time PCR and ELISA assays. Immunogenicity of the recombinant protein was investigated in rabbit by subcutaneous injection. Results indicated that the transgenic plant extract can induce immune responses in the host as confirmed by presence of specific antibodies in the sera in ELISA assay. Western blot assays showed that the foreign gene was actually expressed in transgenic seedlings. CONCLUSIONS: The results obtained in this research provide further evidence on applicability of plant-based recombinant vaccines for protection of poultry against Newcastle disease.
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BACKGROUND: It is well documented that Silver Nanoparticles (SNPs) are potent antimicrobial agents. However, little is known about antimicrobial effects of biologically synthesized SNPs at molecular level. In the present study, efficacy of the green microalgae Chlorella vulgaris in biosynthesis of silver nanoparticles and inhibitory effect of the biosynthesized SNPs on growth and virulence of Staphylococcus aureus (S. aureus) were investigated. METHODS: Algal suspension was incubated in the presence of silver nitrate to induce formation of nanoparticles. The experiment was conducted under a pH range to evaluate pH effect on the shape and properties of nanoparticles. Characterization was performed by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy Dispersive Spectrometry (EDS) and X-ray diffraction analysis (XRD). Moreover, concentration of biosynthesized SNPs was measured by high resolution ICP-OES spectrometer. Antibacterial effect of SNPs on growth of S. aureus was evaluated by broth micro-dilution method. Inhibitory effect of SNPs on alpha hemolysin, a well-known virulence factor of S. aureus was investigated through real time PCR assay. RESULTS: Spherical SNPs were produced with characteristic monodispersity at low and neutral pHs; however, in alkaline condition, nanorod structures were formed. SNPs inhibited growth of S. aureus at concentration of 50 µg/ml. Alpha hemolysin expression was also effectively inhibited by SNPs treatment. CONCLUSION: In general, results revealed formation of spherical silver nanoparticles with inhibitory effects on bacterial growth and antagonist activity on the expression of alpha hemolysin. Moreover, increase in pH to basic condition resulted in aggregation of nanoparticles and formation of rod-like nanostructures.
