Association of AIRE polymorphism and the susceptibility to multiple sclerosis in Iranian population

Background: Multiple Sclerosis [MS] is the most common cause of neurologic disability in young adults. Recently, the AIRE gene was identified as a genetic risk factor for several autoimmune diseases in genome wide association studies. The aim of this study was to further investigate the possible role of the AIRE gene in susceptibility to MS in Iranian population

Methods: A total of 112 MS patients and 94 ethnically matched controls were included in the study. The Single-Nucleotide Polymorphism [SNP] [rs1800520, C>G] with a global MAF=0.2282/1143 was selected and genotyped using HRM real-time PCR method

Results: Results showed that AIRE SNP rs1800520 was significantly less common in the MS patients than in healthy controls [17.8 vs. 28.7%, pc=0.032, OR=0.54,95% CI 0.279,1.042]. Also, the frequency of allele G was significantly higher among the control group than in the case group [37.77 vs. 25%, pc=0.014]. Interestingly, mRNA transcribed on the rs1800520 SNP showed decreased free energy than the wild type suggesting that its increased stability may be responsible for the different activities of the polymorphic AIRE molecule

Conclusions: This is the first study investigating the relationship between AIRE gene and the susceptibility to MS. These results indicated that the rs1800520 SNP is not a susceptibility gene variant for the development of MS in Iranian population

model to study the phenotypic changes of insect cell transfection by copepod super green fluorescent protein [cop-GFP] in baculovirus expression system

Background: Baculovirus expression system is one of the most attractive and powerful eukaryotic expression systems for the production of recombinant proteins. The presence of a biomarker is required to monitor transfection efficiency or protein expression levels in insect cells

Methods: The aim of this study was to construct a baculovirus expression vector encoding a copepod super green fluorescent protein [copGFP]. In this light, the resultant vector was constructed and used for transfection of Spodoptera frugiperda cells

Results: Expression of the copGFP protein in insect cells was confirmed by fluorescent microscopy and Western-blot analysis

Conclusion: The application of copGFP control bacmid can be considered as an appropriate control for insect cell transfection

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

OBJECTIVE@#To express human vascular endothelial growth factor121 (VEGF121) in insect cells.@*METHODS@#A gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF.@*RESULTS@#Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells.@*CONCLUSIONS@#Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective To express human vascular endothelial growth factor121 (VEGF121) in insect cells. Methods A gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

HESA-A exerts its cytoprotective effects through scavenging of free radicals: an in vitro study

Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect. Chinese hamster ovary [CHO] and human embryonic kidney [HEK293T] cells were treated with different concentrations of HESA-A and H2O2 followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit. The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H2O2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H2O2, respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies

Optimization of the expression of genes encoding poly [3-hydroxyalkanoate] synthase from pseudomonas aeruginosa PTCC 1310 in escherichia coli

Over the years, the use of plastics has complicated the problem of disposal of solid wastes. One strategy to reduce plastic waste is the use of biodegradable plastics. A group of these plastics are polyhydroxyalkanoates [PHAs]. To date more than 250 different microorganisms are known to synthesize and accumulate PHA. Most Pseudomonas strains are able to accumulate mcl-PHA. In previous studies, the phaC1 and phaC2 genes were identified in Pseudomonas aeruginosa [P.aeruginosa] PTCC 1310 and were cloned. The aim of this study was to express these genes and optimize the conditions for their expression. The inserts obtained from vectors pTZPHAC1 and pTZPHAC2 were subcloned into pET15b expression vector. After transformation of competent Escherichia coli [E.coli] BL21 [DE3] cells with recombinant plasmids, expression was induced using IPTG. By changing expression conditions such as IPTG concentration, time and temperature of incubation with IPTG, the expression conditions for these enzymes were optimized, and the obtained results were compared using proper statistical analysis. The PHA synthase genes were induced with IPTG and the expressed 62 kDa protein was observed and purified. By changing expression conditions, 1 mM IPTG, 37°C and a 2 hr incubation provided the highest level of protein production in E.coli cells. These results suggest that induction condition of PhaC genes can influence expression of PHA synthase enzymes

Genetically engineered mesenchymal stem cells stably expressing green fluorescent protein

Mesenchymal stem cells [MSCs] are nonhematopoietic stromal cells that are capable of differentiating into and contribute to the regeneration of mesenchymal tissues. Human mesenchymal stem cells [liMSCs] are ideal targets in cell transplantation and tissue engineering. Enhanced green fluorescent protein [EGFP] has been an important reporter gene for gene therapy. The aim of this study was establishment of MSCs expressing GFP. MSCs were isolated and characterized by Immunophenotyping. The pEGFP-Nl plasmid was extracted from previously transformed Escherichia, coli cells and transfected into MSCs using FuGENE HD transfection reagent. Stable cells were established in the presence of geneticin. Expression of GFP was detected by RT-PCR, western blot analysis and immunoflorecent microscope. MSCs were successfully isolated and characterized. The MSCs transfected with the pEGFP-Nl plasmid expressed GFP both in mRNA and protein levels while cells transfected with empty vector did not. The results suggested that this engineered cell line will be used in the future studies and can easily be traced in vivo