HA1 antigen is the major host protective immunogen of the highly pathogenic H5N1 avian influenza A virus (AIV), which leads to great pandemics and epidemics throughout the world. Plants are now gaining widespread acceptance as a general platform for the large scale production of recombinant proteins like influenza vaccines. In present study HA1 antigen of H5N1 was expressed in Alfalfa (Medicago sativa), soybean (Glycine max) and Lettuce (Lactuca sativa) leaves using agro-infiltration. Also the effect of three signal peptide including KDEL, Extensin and ZERA which cause protein accumulation respectively in endoplasmic reticulum, apoplastic space and protein bodies, were analysed. 72 hours after the agro-infiltration, qRT-PCR results showed that lettuce and soybean respectively had the maximum and minimum of HA1 transcripts. The expression of HA1 was detected in leaves extracts by ELISA method. The highest expression was detected in alfalfa leaves with apoplastic signal peptide while the lowest expression was found in lettuce despite the highest transcription rate. The results showed that using agro-infiltration system in alfalfa plant with apoplastic signal peptide was the appropriate strategy for HA1 antigen production in the assayed plants.

The use of plant Oleosin gene is a recommended method for large-scale, easier and cheaper production of recombinant protein. Attachment of oleosin gene to target gene cause to accumulate recombinant protein on seed oil bodies surface. Recent researches indicate that application of tandem Oleosin Genes (polyoleosin) fused to the target gene to facilitate recombinant protein purification is more efficient than single Oleosin . In present research, we designed a synthetic fusion fragment containing 5´- Kozak sequence - His-tags, three Oleosin Genes, a proteolytic site for a peptidase enzyme, Proinsulin protein, and a tetranucleotide stop codon. This synthetic sequence was cloned in binary vector pBI121by enzymatic digestion and ligation method. Then for transformation of cotyledon and hypocotyl explants of Canola, confirmed recombinant construct is transformed to Agrobacterium LBA4404 strain. Probable transgenic shoots were regenerated on selective medium containing kanamycin after 4-6 weeks. The analysis of transgenic shoots at DNA level was performed by PCR and amplification of a 120-bp fragment indicate integration of fusion gene in transgenic plant genome. Finally, the Proinsulin gene transcription was also confirmed by RT- PCR.

protein aggregates generated by different reason, cause many diseases in humans and other organisms. Finding proper ways to stabilize or prevent of protein aggregation could be important. H. sabdariffa is a good source of natural antioxidants which may protect the body from damage effeccted by free radicals. This study examined the effect of aqueous extract of H. sabdariffa on the aggregation of reduced α-lactalbumin. The chaperone properties of H. sabdariffa extract on protein aggregation were determined by measuring light scattering absorption, thioflavin T binding assay, fluorescence, and circular dichroism (CD) spectroscopy. Visible absorption spectroscopy and ThT fluorescence results shows that H. sabdariffa extract prevent protein aggregation and amyloid formation in a concentration-dependent manner. According to the results of Fluorescence Spectroscopy, H. sabdariffa extract interact with hydrophobic area of protein and inhibit aggregation. CD spectroscopy results shows that DTT caused changes in secondary structure of protein and in the presence of extract, fewer changes observed. Our finding suggests the possibility of using H. sabdariffa extract as a potential agent for inhibition of protein aggregation in various disease

To study the mechanisms involved in amyloid formation processes, we made a mammalian cell culture model of amylin aggregation and characterized its properties. Amyloid fibrils were extracted from CHO cells and binding affinity to Thioflavin T and Congo red was investigated. Then the apple-green birefringence of extracted fibrils was detected under polarized light to confirm the presence of aggregated protein in the extracts. To better investigate amyloid formation in CHO cells, we decided to overexpress an amyloidogenic protein in these cells. To do so, we amplified ProIAPP gene and subcloned it in EGFP-N1 expression vector. Then, CHO cells were transfected with EGFP-N1-ProIAPP and EGFP-N1 as a control. The phenotypes of around 100 transfected cells were characterized for several days after the transfection, using fluorescence microscopy. Additionally, the presence of amyloid structure in these cells was detected by Congo red staining under exposure to polarized light. Cell viability assay was performed using Trypan blue staining. Low level natural amyloid was detected in CHO cells. In addition, the ProIAPP-EGFP transfected cells exhibited aggregated phenotype in which the cells with round morphology had far more aggregates than oval ones. We found amyloid fibrils in CHO cells at low level. Overexpression of amylin protein in CHO cells caused aggregation phenotypes. These cells can be used as a model of cell culture for studying protein aggregation. Amyloidogenic properties of this protein could immensely help us to study the mechanisms that are involved in amyloid formation in mammalians including humans.

Some important Strategies to increase recombinant protein yield in plants include expression of protein in suitable plant tissue and improvement of protein stability and accumulation by targeting into subcellular organs using specific targeting signals. The ER contain different molecular chaperons and isomerase for folding of nascent proteins، which can prevent aggregation and formation of incorrect three dimensional structure and promote correct protein folding leading to recombinant protein stability and accumulation. In this perspective، seed-based platforms are particularly attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass، which is beneficial for extraction and downstream processing. In this research for human Gamma interferon expression in Brassica napus seeds and targeting into ER، we used seed specific promoter (Napin) and C-terminal KDEL sequence (ER retention signal). The fragment was cloned into pGEM®-T Easy vector and then was confirmed by PCR، restriction enzyme analysis and sequencing. The authentic PCR fragment was subcloned into a plant binary vector (pBI121). This construct cassette (pBI IFNγ) was transferred to A. tumefaciens LBA4404 and then used to transform B. napus using an Agrobacterium-mediated petiole cotyledonary transformation. The transformed plants were screened on antibiotic-contained media The presence and expression of the transgene was confirmed in the transformants by PCR and SDS-PAGE. ELISA test was used for biological activity.

Crocin, an important saffron ingredient, showed anticancer activity in a variety of cancer types, particularly breast cancer. However, little information is available on the mechanism of its action. Previous studies indicate apoptosis induction by crocin in some cancer cells. This study aims to investigate the effect of crocin on the MDA-MB-468 breast cancer cell line in order to investigate its effect on caspase 9 (Cas9) and cleaved-Cas9, expression and splicing of XBP1, and accumulation of LC3-II.
We used the MTT assay to investigate the cytotoxic effect of crocin on MDA-MB-468 breast cancer cells. Next, Cas9 and cleaved-Cas9 levels were evaluated by Western blot analysis. Splicing of XBP1 mRNA and expression of the spliced protein (XBP1s) was investigated by RT-PCR and Western blot, respectively. The accumulation of LC3-II was also evaluated by Western blot. The obtained results were analyzed and reported by Image J software.
The results showed a time and dose-dependent cytotoxic effect of crocin in MDA-MB-468 cells. The expression of Cas9 and its cleavage, therefore, the ratio of cleaved-Cas9/Cas9 significantly increased. Crocin treatment led to a noticeable increase in splicing of XBP1 mRNA, expression of XBP1s, accumulation of LC3-II, and increased the LC3-II/LC3-I ratio in these cells.
The data have shown induction of apoptosis in these breast cancer cell lines after crocin treatment. Because of the observed changes in UPR markers and autophagy, it seems that these pathways are possibly involved in this process and in intracellular regulations.

Plant diseases are the most critical limitation factors in agricultural products. Citrus bacterial blast disease caused by Pseudomonas syringae pv. syringae is one of the most common diseases in in citrus gardens except in tropical region of the world. Host plants interact to bacterial pathogenes by inducing of resistance genes. Accumulation of pathogenesis related proteins such as, chitinase, ß-1, 3 and some of the other genes like phenyl alanine ammonia lyase and transcriptional factor WRKY40 are among the defens mechanisms in challengins to pathogens. In this research, the transcript levels of PR2, PR3, PAL and WRKY40 genes were evaluated in sour orange, limequat and okitsu lines in stimulating with Pss using quantitative real time PCR technique at different time courses. After inoculation of citrus lines to Pss, total RNA were extracted from samples at different time courses. Complementary DNA were synthesized and the expression levels of mentioned genes evaluated. Result of this study showed that transcripts levels of PR2, PR3, PAL and WRKY40 genes increased faster and significantly higher in okitsu and sour orange than limequat.

The production of pharmaceutical compounds having role in treating diseases like AIDS is necessary. In this regard, griffithsin (GRFT) (an algae lectin) can inhibit HIV entry to the cell at picomolar concentration. To production of recombinant GRFT protein, alfalfa, soybean and lettuce were used as hosts for transient expression. Effect of KDEL, signal peptide of carrot extensin and SP subunit of ZERA signal peptide that target protein to endoplasmic reticulum, apoplastic space and space around the cell wall, respectively was studied. 72 hours after agroinfiltration, qRT- PCR analysis showed high potential of lettuce to express of GRFT at transcript level while alfalfa had lowest transcription level. ELISA result using polyclonal anti-mouse anti-GRFT antibody confirmed that targeting of GRFT to apoplast space of soybean led to highest amount of GRFT. Lettuce despite of high level of gene expression, showed low level of GRFT accumulation which may be due to posttranscriptional and post translational modifications. Our results suggest using of soybean leaf tissue for fast and reliable production of pharmaceutical using transient expression.

This study was performed to investigate the effect of methyl jasmonate (MeJA) elicitor on green fluorescent protein (GFP) and Anti vascular endothelial growth factor (Anti-VEGFR2) proteins accumulation. A factorial experiment based on completely randomized design (CRD) was performed primarily to evaluate the effect of MeJA and days after inoculation on GFP expression in Brassica napus and Nicotiana benthamiana. RT-PCR and qRt-PCR assay was performed for GFP gene transcription confirmation at mRNA level. ELISA was done to investigate effect of the elicitor on GFP and Anti-VEGFR2 accumulation. The optimum treatment combination was then used to check Anti-VEGFR2 accumulation. GFP expression was significantly enhanced using M1D10 combination (the best treatment combination) in the two plants. This increase was greater in Brassica napus than in Nicotiana benthamiana. GFP and Anti-VEGFR2 transcript levels showed no significant differences between the optimized and control treatments in the qRT-PCR experiment, but the quantity of Anti-VEGFR2 protein accumulated in optimized treatments was higher than in control. A simple explanation for the improved accumulation of GFP and Anti-VEGFR2 would be a low ‘sink pressure’ on amino acid pools towards RuBisCO biosynthesis in RuBisCO-depleted leaves, and a resulting increased availability of metabolite and cellular resources for the production of less abundant (e.g., recombinant) proteins. This research showed that MeJA elicitor increased the accumulation of both proteins.

Cereal yellow dwarf viruses are among the most destructive cereal viruses worldwide. In this study, subcellular localizations of silencing suppressor proteins ( P0) from two cereal yellow dwarf viruses, Cereal yellow dwarf virus-RPV (CYDV-RPV) and Cereal yellow dwarf virus-RPS (CYDV-RPS), were examined via confocal microscopy. To do so, P0 genes were synthesized by polymerase chain reaction (PCR) and were cloned through Gateway cloning system. Agrobacterium. tumefaciens clones harboring GFP:P0 constructs were infiltrated into Nicotiana benthamiana plants. Transient expression of P0 proteins with N-terminal GPF fusion enabled us to examine their subcellular localizations in plant cells, 24 hours post infiltration. Results showed that both proteins are nuclear-cytoplasmic but different tendencies were observed in their localizations. In other words, P0 of CYDV-RPS was more nuclear oriented than P0 of CYDV-RPV. In the Western blot analysis using GFP polyclonal antibody only GFP:P0 proteins but no free GFP, were detected indicating that localizations were related to GFP:P0 proteins.