Prediction of Brucella omp25 and BLS antigens epitopes and their interaction with MHC molecules in sheep by in silico methods (original) (raw)
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Expression, Purfication and Endotoxin Removal of Brucella Melitensis Dnak and OMP31 Proteins
ISMJ, 2014
Background: New strategies are needed to protect against Brucella melitensis infection. Subunit vaccines offer a promising approach because they can stimulate both cellular and humoral immunity, so high production of recombinant protein with less content of endotoxin is desired. In present study, we described a method for expression and purification of B.melitensis recombinant DnaK(rDnaK) and Omp31(rOmp31) proteins while less content of endotoxins were detected in final product. Material and Methods: Recombinant pET-dnak and pDEST-omp31 plasmids were transformed into competent expression host E.coli BL21 (DE3). After induction by IPTG, bacteria were grown at 20 • C for 22h. Then recombinant proteins were purified by Ni-NTA Agarose. Purification was done while two methods using Triton X-114 in washing steps and standard protocol (without detergent) were used in parallel. Results: rDnak and rOmp31 were purified by using Urea. We could obtain 20 and 8 mg recombinant proteins from rDnak and rOmp31 from 1 liter medium, respectively. The amount of endotoxins in final products was less content of 0.05 EU/mg. Furthermore, recovery of protein was up to 80% as compared to the standard protocol. Conclusion: The method used in this study, gives a product with very low extent of endotoxin, but 20% of recombinant proteins were lost. So we think the method described here can be used for purification and endotoxin removal of other recombinant proteins with similar physiologic properties.
Design of Optimized PCR Method for Detection of SHV Type ESBL Genes
2016
Introduction: ESBL is an important factor in antibiotic resistance. Antibiotic resistance has been proposed as a global problem in recent decades and it causes many deaths all over the world; therefore, rapid and accurate diagnosis can be major step in solving this problem. This study aimed to identify SHV-type gene with PCR method. Methods and Materials: SHV gene sequences were extracted and aligned from Genbank. Specific PCR primers were designed according to the consensus of the SHV genes. A plasmid containing positive control of the gene was created by using PCR and cloning methods. The assay specificity was evaluated using Brucella genomic DNA and a panel containing genomes of 10 gram-positive and gram-negative organisms. The PCR assay was highly specific and no amplification products were observed from the non-SHV organisms, an other test of the specifity was done by the use of CTX-M and TEM betalactamases. Results: A band with a the length of 199bp was indicated in the agarose gel electrophoresis of the PCR product in accordance with the desired sequence. Showing the same fragment in PCR confirmed the recombinant plasmid of coloning product containing the SHV gene. When the plasmid was subjected to the SHV-PCR, a ladder-like pattern was visualized on the agarose gel. The pattern has never observed in the case of negative controls.The results of specify assay conveyed that primers were separately for SHV gene. Discussion and Conclusions: The results of this study indicated that PCR assay is a simple, rapid, sensitive and specific technique for detection of SHV gene that may improve diagnostic potential in clinical laboratories.
New Cellular and Molecular Biotechnology Journal, 2019
Aim and Background: Klebsiella pneumoniae is a common cause of β-lactamase-associated infections in hospitals. The present study aimed to determine the frequency of antibiotic resistance genes in Klebsiella pneumoniae strains producing IMP-1 and TEM β-lactamase. Materials and methods: The present research identified 94 samples of K. pneumoniae, using antibiogram for the phenotypic confirmation of ESBLs. The antibiotic resistance of the isolates and the prevalence of TEM and IMP-1 genes were determined using PCR method. Findings: Of 94 samples, 77.6% were ESBL-positive and 22.3% ESBL-negative. A total of 4.1% of the samples carried the IMP-1 gene and 43.8% the TEM gene, while 43.8% of the samples carried both genes. Conclusion: Given that TEM and IMP-1 genes were commonly present in a large number of the resistant samples, physicians are recommended to use therapeutic measures properly, and to prescribe antibiotics rationally.
Quarterly of the Horizon of Medical Sciences
Aims Programmed cell death protein-1 (PD-1) is a membrane receptor expressed on the surface of T and B lymphocytes, monocytes, natural killers, and dendritic cells. In cancer, the PD-1/PD-L1 system prevents the proliferation of T lymphocytes and causes the release of cytokines and cytotoxicity, which leads to the apoptosis of tumor-specific T cells, thereby preventing the immune response to cancer cells. Methods & Materials In this study, the extracellular part of the humanized PD-1 protein was cloned and expressed, and the protein was injected as an antigen into a camel (Camelus dromedarius) to obtain a camel polyclonal antibody against PD-1 protein. Findings The obtained results indicate the proper expression of the protein in the prokaryotic system. Also, using various tests, such as ELISA and western blot, it was confirmed that the polyclonal antibody obtained from camel can identify PD-1 protein. Conclusion This study showed that because of the advantages, such as the ability t...
Journal of Mazandaran University of Medical Sciences, 2016
Background and purpose: Platelets communicate with different immune cells and can activate B-lymphocytes and induce the production of antibodies from these cells. Platelet microparticles (MPs) originate from platelets and express the surface markers of platelets. This study aimed at investigating the ability of these microvesicles on production of antibodies from B-lymphocytes. Materials and methods: In this experimental study, platelet MPs were isolated from platelet concentrates and B cells were isolated from human whole blood. Then MPs were co-cultured with Blymphocytes. In different days of culture, the production of IgG antibodies was studied in the supernatants of culture medium using ELISA method. The results were analyzed by paired-samples t-test. Pvalue < 0.05 was considered statistically significant. Results: Platelet MPs stimulate the production of antibodies by B-lymphocytes. During 5-day coculture, significant increase was observed in the production of IgG antibodies...
Research on Animal Production
Introduction and Objective: In the dairy industry, it is important to identify the genotypes resistant to mastitis while select high-yielding dairy cows. Bovine β-difensin gene polymorphism can be used as a molecular marker in the selection of mastitis-resistant dairy cows. The aim of this study was to investigate the relationship between point mutation of β4difensin gene and the occurrence of clinical mastitis in the dairy cows using RFLP-PCR method and identification of superior genotype of dairy cows β-difensin gene for mastitis resistance. Material and Methods: blood sample (with EDTA anticoagulant) was taken from total 73 cows (32 with a history of mastitis and 41 with no history of mastitis) in an industrial dairy farm. After DNA extraction amplification of the region of bovine β-difensin gene (393 bp) was performed. PCR products were digested by endonuclease (NlaIII (Hin1II)) for rapid detection of polymorphism in the 2239 region of the β-difensin gene (C to T conversion). Statistical analysis was performed by Chi-square test. Results: Allele frequencies were 0.68 and 0.32 for C and T, respectively. Statistical analysis was performed by Chi-square test. The results of this study indicate the association between number of mastitis and polymorphism in the β-difensin gene. In other words, the incidence of mastitis was numerically lower in cows with T allele than in cows without it (p=0.07). Conclusion: It can be concluded that the bovine β-difensin gene can also be used as a molecular marker in the selection of dairy cows to reduce the incidence of mastitis.
Journal of Entomological Society of Iran, 2020
The cotton bollworm, Helicoverpa armigera (Hubner),is a wide host range pest that causes severe economic damages to agricultural crops in Iran and all around the world. During recent years, chemical insecticides have been used as the most effective strategy in control of this pest, but due to their hazardous effects, most of the researches are being conducted to offer an alternative approach for chemical control. In this regard, digestive systems, in particular inhibition of insect digestive enzymes, are considered as a target for pest control. Here, we used the original pro-region of H. armigera chymotrypsin as a potent and specific inhibitor of the pest enzyme. The structural model of the insect chymotrypsin was predicted based on homology modeling and the crystal structure of Bos taurus L. as template. The reliability of the model was assessed using VERIFY_3D, ERRAT, PROCHECK, WHAT-IF and Z-scores, and the results confirmed that the predicted structural model has an appropriate q...