Showing 15 results for Gene Expression
Hamid Abtahi, Ali Hatef Salmanian, Sima Rafati, Ghorban Behzadian Nejad,
Volume 7, Issue 1 (3-2004)
Abstract
Introduction: Brucellosis is one of the most important zoonotic diseases that causes miscarriage and infertility in animals and causes human fever. The use of the common SS9 strain of Brucella abortus has several side effects for livestock. Brucella P39 protein is one of the plasma peripheral space proteins that is considered as one of the important immunogenic indicators. With the production of the new protein combination of P39, more studies can be done on the ability of this protein to stimulate immune responses against Brucella. Therefore, in this research, the production and purification of this protein in Escherichia coli bacteria has been done as a new compound.
method: In this experimental study, using the polymerase chain reaction, the P39 gene was propagated by the bacterium Brucella abortus. After purifying the P39 gene, it was cloned into plasmid carriers pSK+ and pGEX4T1. Therefore, pSK+-P39 and pGEX4T1-P39 structures were prepared. To produce the recombinant protein P39, the plasmid structure pGEX4T1-P39 first entered the Escherichia coli bacterium BL21. The protein was then produced by IPTG by induction of pGEX4T1-P39 plasmid. The resulting protein was purified using the orderly purification protein glutathione S-transferase. The amount of purified protein was measured using the Brad Ford method.
Results: The nucleotide sequence of the gene propagated by the cloned PCR in the plasmid carrier pSK+ was exactly the same as the P39 gene of Brucella abortus. Production of P39 protein was performed by induction of pGEX4T1-P39 plasmid. The purified protein content was 200 micrograms per milliliter.
Conclusion: The production of the new protein P39 compound Brucella Abortus, which is unstable in the cytoplasm of the Escherichia coli bacterium, is possible using carriers with additive proteins such as pGEX4T1 in the host of Escherichia coli strain BL21.
Hamid Abtahi, Ghasem Mosayebi, Ali Hatef Soleimanian,
Volume 10, Issue 3 (6-2007)
Abstract
Introduction: Streptolysin O (SLO) is an antigenic protein that is secreted by Streptococcus pyogenes. Streptococcal infections are diagnosed with anti streptolysin O. At present, streptolysin O is produced by vectors that have fusion protein. In this study streptolysin O without fusion protein vectors is produced. Materials and Methods: In this experimental study, Streptolysin O gene was amplified by Polymerase chain reaction (PCR) method and subcloned to prokaryotic expression vector pET28a. Escherichia coli BL21-DE3-plySs were transformed with pET28a-SLO and gene expression was induced by IPTG. Then it was purified by Ni-NTA kit. The concentration of SLO was assayed by Bradford method. To confirm recombinant SLO Western Blot was used. Results: The sequencing result was confirmed by Sanger method and was the same as SLO gene. Escherichia coli BL21 (DE3) pLysS was transformed with pET28a-SLO and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography with Ni-NTA resin. The concentration of purified protein was 100µg/ml. The integrity of product was confirmed by Western Blot analysis using a mouse anti streptolysin O. Conclusion: Data showed that recombinant SLO protein can be produced by pET28a in Escherichia coli. This protein maintains its antigenic effect very well. Therefore, recombinant SLO has same epitopes with natural form of this antigen.
Mahmoud Kamani, Hamid Abtahi, Ghasem Mosayebi, Razieh Nazari, Masode Karimi,
Volume 14, Issue 1 (3-2011)
Abstract
Background: In pyoderma infections, the density of pus is related to desoxiribo-nucleoproteins. The use of streptodornase (DNase) in combination with streptokinase can help dissolve purulent secretions of infections which results in healing the wound through the discharge of pus from the necrotic tissue. The aim of this study was to produce recombinant streptodornase from group A strain of Streptococcus pyogenes which is highly efficient in terms of active streptodornase production using expression vector. Materials and Methods: In this applied-fundamental study, genomic DNA of streptodornase gene (sd) was extracted by phenol-chloroform. Then by using specific primers of streptodornase gene, it was amplified through PCR. The resulting streptodornase gene was cloned in pGEX4T1-sd transformer for expression and the pGEX4T1-sd plasmid was transferred to the sd. E.coli BL21. Protein production was done by induction via IPTG and optimization of the conditions. The recombinant protein was purified using the glutathione sepharose 4B kit. Results: The nucleotide sequence of PCR and group A streptodornase Streptococcus was totally the same. The production of the streptodornase recombinant protein was done by inducing pGEX4T1-sd plasmid via Isopropyl β-D-1-thiogalactopyranoside. Protein purification was done through affinity-chromatography by using glutathione sepharose 4B. The recombinant protein was reacted with anti-streptodornase mouse serum through Western-Blot method. Conclusion: Recombinant streptodornase can be produced by pGEX4T1 in E. coli. The recombinant protein maintains its antigenic property desirably. Noticing the domestic need in Iran, low rate of production, and pathogenesis of streptococci, production of this recombinant product is feasible.
Azar Moradkhani, Hamid Abtahi, Iraj Pakzad, Masode Karimi,
Volume 14, Issue 2 (5-2011)
Abstract
Background: Hyaluronidase A is an antigenic protein that is secreted by Streptococcus pyogenes. Nowadays, streptococcal infections are diagnosed by tracking down anti-hyaluronidase A antibodies. In this study, the attempt was made to generate recombinant hyaluronidase A in E. coli.
Materials and Methods: In this experimental study, through designing specific primers and polymerase chain reaction (PCR), hyaluronidase A gene was amplified and after purification, it was sub-cloned in plasmid expression vector pET32a. Then pET32a-hylA was transferred to E. coli BL21-DE3-plySs. Protein generation induced by IPTG. The recombinant protein was purified by Ni-NTA kit and its concentration was assayed by Bradford method. Western-Blot analysis was run for verifying the recombinant hyaluronidase A.
Results: The nucleotide sequencing of the gene amplified by PCR was the same as hyaluronidase A gene from Streptococcus pyogenes. Production of the recombinant hyaluronidase A via induction by pET32a-hylA plasmid was done through IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. The concentration of purified protein was 500µg/ml. analysis using a mouse anti-hyaluronidase A serum was reacted with the generated protein using Western-Blot analysis.
Conclusion: Recombinant HylA protein can be generated in E.coli and the resulting protein maintains its antigenic properties desirably.
Neda Molaee, Hamid Abtahi,
Volume 14, Issue 4 (9-2011)
Abstract
Background: Streptokinase is one of the antigenic proteins secreted by streptococcus pyogenes. This protein has an important role in bacterial pathogenesis. The aim of this study was to produce recombinant forms of this enzyme so that the product would change in accordance with changes in the media.
Materials and Methods: In this experimental study, we amplified the streptokinase gene by polymerase chain reaction (PCR) method. After extraction, it was sub-cloned to prokaryotic expression vector pET32a. pET32a-Ska was transferred to E.coli BL21-DE3-plySs strain. Protein production was induced by IPTG and optimization of culture media and OD of bacteria. The recombinant protein was extracted by Ni-NTA and its concentration was measured by Bradford assay. Western- Blot analysis was used to verify the recombinant protein.
Results: The nucleotide sequence of the amplified gene was the same as streptokinase gene of the streptococcus pyogenes. The production of recombinant streptokinase by induction of plasmid pET32a-Ska was done by IPTG. The recombinant streptokinase had the same antigenic properties as natural streptokinase. The largest amount of recombinant protein was produced in bacteria concentrations with OD = 0.8. Also, the production of the recombinant protein was higher in media with no glucose.
Conclusion: Changes in culture media can increase the production of recombinant proteins in host bacteria. The presence of nutrients, such as glucose, alone not only can not increase the amount of production but it might even decrease it
Hamid Kazemian, Mohammad Najafi-Mosleh, Hamid Abtahi,
Volume 15, Issue 7 (12-2012)
Abstract
Background: Vibrio cholera is an important agent causing cholera in human. The expression of Flagellum and the movement of the bacterium are critical in the colonization and virulence of Vibrio cholera. FlaA gene is one the five genes encoding Flagellin which plays an important role in the activity and movement of the bacterium and its colonization which has a significant role in its immunogenicity. The aim of this study was to express and produce the recombinant FlaA protein in E.coli using Western blot method. Materials and Methods: In this experimental study, FlaA gene was proliferated by PCR method using the specific primers and cloned with BamHI and Xhol in pTz57R/T. Then it was proliferated and sequenced in DH5a vector of E.coli. The cloned FlaA gene was inserted into pGEX-4T-1 vector. The cloned vector was transformed to BL21-DE3 of E. coli and successfully expressed by induction of IPTG. The expressed protein was purified by GST affinity resin. For preparation of the primary antibody, the purified recombinant protein was injected to rats. Western blot assay method was used for determining the antigenicity of the recombinant FlaA. Results: Determination of gene sequencing showed that this gene has been proliferated properly and the antibody used in Western blot verified the production of the recombinant protein. Conclusion: The results of this study demonstrate that FlaA protein is immunogenic and can be evaluated in vaccine designing and as a diagnostic tool for detection of cholera infection.
Malihe Bahadori, Saedeh Zafar Balanezjad, Mohammad Mahdi Forghanifard ,
Volume 17, Issue 4 (7-2014)
Abstract
Background: According to the important of SALL4 gene during the development of embryonic neurvous system, our aim in this study was to analyze and quantify mRNA expression of SALL4 in Prosencephalon during different stages of chicken embryogenesis.
Materials and Methods: In this experimental study, incubated Ross fertilized eggs were applied in 37°C-37.5°C in 60-65% humidified atmosphere after beginning of embryogenesis. Prosencephalon part of the brain tissue was collected from the eggs, daily. Total RNA extraction and cDNA synthesis was performed from resected tissues. The synthesized cDNA was used as template for quantitatively analysis of SALL4 mRNA expression by real-time PCR.
Results: The Results indicate that the level of SALL4 gene expression is significantly variable during embryogenesis. However it doesn’t show variation during the early days. The maximum copy number of SALL4 mRNA was quantified on 15 th day of chicken development.
Conclusion: SALL4 mRNA expression is high when the Prosencephalon is under development, using of HAMBURGER–HAMILTON chart, there is relation between increasing SALL4 expression and developing limbs and anterior brain.
Nafise O Sadat Mirjamali Mehrabadi, Safieh Soufian, Hamid Abtahi,
Volume 17, Issue 4 (7-2014)
Abstract
Background: Streptococcus pyogenes produce extracellular hyaluronidase enzyme which is directly associated with the spreading of the organism during infection. Hyaluronidase enzyme is able to break hyaluronic acid or interstitial cement. This enzyme might be used in cancer treatment.The objective of the present study was to clone and express the nucleotide sequence of this enzyme which is involved in hyaluronidase enzymatic activity.
Materials and Methods: The enzymatic region of hyaluronidase gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify the region. The amplified product was cloned into the expression vector pET32a. E. coli BL21 (DE3) pLYsS was transformed with recombinant plasmids. Then gene expression was induced by IPTG. The expressed protein was purified successfully via affinity chromatography by NiNTA kit. The integrity of the product was confirmed by western-blot analysis.
Results: The nucleotide sequence of amplified gene was consistent with the streptocuccal hyaluronidase gene. The concentration of recombinant protein calculated to 500 mg purified protein per liter. The enzymatic region of recombinant protein from Streptococcus pyogenes was recognized by all five patient’s sera with Streptococcus infection.
Conclusion: In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in Escherichia coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.
Morteza Sadeghi, Zohreh Hojati, Kamran Ghaedi,
Volume 17, Issue 8 (11-2014)
Abstract
Background: Vascular endothelial growth factor (vegf) is one of the most important regulator of angiogenesis, there are some reports about the relation of VEGF over expression and progression of tumor in several cancers. The aim of this study is assay of four VEGF isoforms expression in breast cancer tumor samples.
Materials and Methods: 25 breast cancer tumor samples and 25 health samples were used in this study, mRNA was extracted from each sample and then cDNA was made. The expression of four isoforms VEGF121, VEGF165, VEGF183 and VEGF189 was measured by real time reverse transcription PCR (RT-PCR) and gel electrophoresis.
Results: Among the four isoforms, VEGF165 and VEGF 121 had maximum and VEGF 183 and VEGF 189 had minimum expression level in all samples. The total expression level of VEGF had a significant increase in tumor samples in comparison with the control samples (4/6, p<0.01).
Conclusion: There is a significant relation between the VEGF over expression and breast cancer tumor formation, which it can be used as a prognosis marker of breast cancer in future.
Raziyeh Kheirjou, Mohammad Hasan Heidari, Mohammad Bayat, Masoumeh Rajabi Bazl, Rasoul Ganji, Abbas Piryaei,
Volume 18, Issue 5 (8-2015)
Abstract
Background: Wound healing is a complex process that is impaired in diabetic patients due to several factors. So far, the positive effects of mesenchymal stem cells secretions in wound healing process have been reported. In this study, we investigated the effect of human mesenchymal stem cells Conditioned media on expression of effective factors involved in wound healing.
Materials and Methods: 27 rats were divided into 5 groups: no wound control, normal control, diabetic control, diabetic placebo and diabetic experimental. Diabetes was induced by Alloxan. A wound was created on the back of the rats. Then, the conditioned medium was prepared from mesenchymal stem cells. Diabetic experimental rats received 200 microliter of conditioned medium intravenously. The wounds were sampled and expression of KGF and TGF-&beta1 genes was examined by RT-PCR on days four and seven after wounding.
Results: In the diabetic experimental group, expression of KGF gene at fourth and seventh days had been non-significantly increased in comparison to diabetic control group. While, expression of TGF-&beta1 gene in diabetic experimental group compared to diabetic control group had been significantly (p<0.05) increased on fourth day, and non-significantly increased on seventh day.
Conclusion: It seems that using the conditioned medium derived from human mesenchymal stem cells positively affects the expression of trophic and inflammatory factors involved in diabetic skin wound healing.
Shirin Abdolvand, Mehdi Moghanibashi, Parisa Mohamadinejad,
Volume 20, Issue 3 (6-2017)
Abstract
Abstract
Background: The incidence of gastric cancer is different in two sexes with ratio 2 to 1 that it is more common in men. The most important biologically reason is sexual hormones between two sexes that lead to sexual dimorphism and in turn can cause a sex bias in incidence of disease between two sexes. Recently, studies have shown that microRNA is involved in sexual dimorphism in gene expression. Given the sexual dimorphism in the incidence of gastric cancer and sex hormones response elements in the regulatory regions of miR-146a and miR-148a genes, in this study, the expression of these two genes in the stomach of healthy men and women at different age groups were compared.
Materials and Methods: Using endoscopy, gastric antrum tissues of 35 healthy women and 35 healthy men were collected. After RNA extraction and synthesis of cDNA, the expression of miR-146a and miR-148a genes were compared between sexes by Real time RT-PCR and data were analyzed using independent sample t and ANOVA tests.
Results: There was no difference between men and women in genes expression of miR-146a and miR-148a. However, expression of miR-146a gene was significantly more in men under 45 years than men over 45 years (p= 0.017, df= 14, t= 1.47). Also, expression of miR-148a gene was significantly more in men over 45 years than men under 45 years (p=0.001, df= 12, t= 1.28). But the expression of both genes had no significant difference between women under 45 years and women over 45 years.
Conclusion: Expression of miR-146a and miR-148a genes in the stomach is increased and decreased with aging in men, respectively.
Mehri Jamilian, Jamilian Somayeh Jamshidi,
Volume 20, Issue 10 (1-2018)
Abstract
Abstract
Background: Selenium supplement has multiple important effects, including anti-inflammatory effect. The aim of this study was to assess the effects of selenium supplement on gene expression of inflammatory cytokines and vascular endothelial growth factor in gestational diabetes.
Materials and Methods: This randomized double blind placebo control trial was performed on 40 patients suffering from GDM aged 18–40 years old. Participants were randomly divided into interventional group receiving 200mg/day selenium supplements (n=20) and control group receiving placebo (n=20) for 6 weeks. Primary outcome was gene expression of inflammatory cytokines and VEGF which were assessed in lymphocyte of GDM patients by RT-PCR method.
Results: After 6 weeks intervention, in comparison with the control group, interventional group showed down regulation of gene expression of tumor necrosis factor alpha (TNF–α) (p=0.02) and transforming growth factor beta (TGF–β) (p=0.01) and up-regulation of gene expression of vascular endothelial (VEGF) (p = 0.03) in lymphocytes of GDM. There was not any significant change following intervention with selenium regarding gene expression of interleukin IL-1 β and IL-8 in lymphocytes of GDM patients.
Conclusion: 6 weeks supplementation with selenium in patients with GDM can cause down regulated gene expression of TNF-α and TGF–β, and up regulated gene expression of VEGF. Selenium supplement had not any effect on gene expression of IL-1 β and IL-8.
Parvin Javdan, Somayeh Reiisi, Parisa Mohammadi Nejad,
Volume 21, Issue 1 (4-2018)
Abstract
Abstract
Background: Ovarian cancer is one of the common malignancies within gynecological cancers. Its lethality may be due to problems in distinguishing it at an early stage and lack of effective managements for patients with a progressive or recurrent status. Therefore, there is an essential need for prognostic biomarkers to diagnose or identifying mechanism of disease for effective treatment. It has been found out that, TRAF4 gene was significantly transformed in different cancers. Therefore, the aim of the present study was to investigate the TRAF4 gene expression in ovarian cancer.
Materials and Methods: In this study, 40 formalin fixed paraffin embedded tumoral tissues of ovarian cancer and 40 non-tumoral tissues were enrolled. Afterwards total RNA extraction and cDNA was synthesized, the relative gene expression was determined using quantitative real-time PCR (qRT-PCR) and evaluated by 2-∆∆ct method. Finally, the expression pattern was analyzed by statistical analysis.
Results: The results of recent study showed that TRAF4 expression was significantly increased in tumoral samples (p=0.0001). According to the study of demographic and clinopathology information with gene expression, there was seen a significant relationship between metastasis and up-regulation of gene. Also, there was a higher expression in TRAF4 gene in patient’s ≤ 48 years old.
Conclusion: According to different studies, it seems that TRAF4 over expression is likely due to amplification of gene copies in chromosomal zone in cancers. Considering the results of present study and the over expression of TRAF4 in ovarian cancer specimen, especially over expression in patients≤48 years old, TRAF4 gene can be considered as a diagnostic biomarker.
Zakiyeh Gharib, Naser Sanchooli, Nima Sanadgol,
Volume 23, Issue 2 (5-2020)
Abstract
Background and Aim: This study aimed to investigate the association between Endoplasmic Reticulum autophagy (ER-phagy) and Alzheimer’s Disease (AD) by analyzing the expression patterns of related genes in animal models.
Methods & Materials: Microarray data of AD patients’ brain tissues were extracted from the Gene Expression Omnibus (GEO) database. These data were first analyzed in GEO2R online tool. Then, the expression of ER-phagy related genes were isolated and the protein interaction networks were plotted by STRING database for the genes with increased expression. Finally, the relationship between the genes that had significant increased expression were designed, and the expression of new identified genes in each study was examined.
Ethical Considerations: All ethical principles were considered in this article.
Results: Genes involved in ER-phagy showed a sporadic expression in different AD models. An increase in the expression of ER-phagy regulatory 1 (FAM134B) gene was observed in studies with the mutation in both Microtubule-associated Protein Tau (MAPT) and Amyloid Precursor Protein (APP) genes. Increase in the expression of NPC intracellular cholesterol transporter 1 (NPC1) gene was observed in two studies that had mutations in APP, Presenilin 1 (PSEN1) and MAPT genes. Moreover, SEC62 homolog and Cell Cycle Progression 1 (CCPG1) genes both showed decreased expression in one study. Finally, the expression of Reticulon 3 (RTN3) was not significant in any of the studies.
Conclusion: The genes involved in ER-phagy have a sporadic expression in AD models, where only two genes FAM134B and NPC1 are involved in AD. The FAM134B gene seems to interact with the Wnk1 gene, which plays a role in cell survival and proliferation, in the hippocampus and forebrain. It also interacts with the Map1lc3b gene, which has a role in phagosome deletion and protein ubiquitination, in the forebrain. It also interacts with the Map1lc3b gene, which has a role in phagosome deletion and protein ubiquitination, in the forebrain. NPC1 had interaction with the Abcg1 gene, which activates lipid homeostasis, in the subventricular zone.
Hamid Moghavemi, Sadegh Abbasian, Mohammad Ali Sardar,
Volume 27, Issue 5 (12-2024)
Abstract
Introduction: Reducing physical activity as well as consuming more calories than the body needs increases obesity and its related disorders, such as metabolic syndrome. Therefore, this study aimed to determine the effect of eight weeks of high-intensity interval training (HIIT) and resistance training (RT) on brain tissue gene expression of AKT2 and insulin resistance in obese Wistar rats.
Methods: 30 male rats weighing 160 to 185 grams were fed a high-fat diet for 12 weeks. After the approval of the obesity protocol of increasing the weight of the rats, which to be more than 300 grams, the rats were divided into three groups, including the control group (n = 10), HIIT group (n = 10), as well as RT group (n = 10). Until the end, the rats continued to eat a high-fat diet. HIIT was performed for eight weeks and five sessions per week, with an intensity of 80 to 95% of maximum oxygen consumption on a treadmill. Moreover, RT was performed with an intensity of 40-60% of a maximum repetition on the ladder. After eight weeks of training interventions, the expression level of the AKT2 gene in brain tissue was measured by the real-time PCR method.
Results: The results of the present study demonstrated a significant increase in AKT2 gene expression of HIIT and RT groups compared to the control group (P < 0.05). Furthermore, the results illustrated that the insulin resistance of rats in both training groups was significantly reduced (P < 0.05).
Conclusions: According to the findings of the present research, it could be concluded that HIIT, as well as RT interventions, probably causes an increase in AKT2 gene expression and could be effective in reducing insulin resistance and improving glucose profile.
