Selection and characterization of single-stranded DNA aptamers against interleukin-5

Asthma as a chronic inflammatory disorder is associated with many cytokines like interleukin-5 (IL-5) which plays essential role in eosinophil differentiation and maturation. Accordingly, blockage of IL-5 using mepalizumab has been considered as a promising therapeutic approach for asthma. Despite the monocolonal antibody advantages, some restrictions provided an acceptable background for alternative agents like aptamers which could replace with antibodies. In the current study, aptamer isolation against IL-5 molecule was intended, according to the valuable benefits of aptamers over antibodies. HEK-293T/IL-5 cell was constructed to select aptamer using cell-systematic evolution of ligands by exponential enrichment (SELEX) method. Integration of the IL-5 fragment to genome of the HEK-293T was verified by polymerase chain reaction on the genomic DNA of the transfected cells. Moreover, IL-5 protein expression on the cell surface was confirmed using flow cytometry analysis. Then, cell SELEX was carried out in 12 rounds and isolated aptamers were evaluated by flow cytometry analysis. The selected clones were then sequenced and assessed for any possible secondary structure. The results of this study led to the selection of 19 different single-stranded DNA clones after 12 rounds of selection which were clustered to five groups based on common structural motifs. In conclusion, the findings revealed the isolation of IL-5-specific single-stranded DNA aptamers, which can further be substituted with mepolizumab

Optimization of interleukin-5 protein expression in BL21 strain of Escherichia coli

Background: In asthma, the relationship between number of eosinophils and severity of this disease supports the hypothesis that eosinophil is the major effector cell in inflammation of airway. Evolution of eosinophils is regulated by interlukin-5 (IL-5). Therefore, by blocking IL-5, at least one major reason of asthma would be prevented. To produce antagonists against IL-5 (like aptamer), it is necessary to have this protein in large scale and high purity. This study aimed to optimize IL-5 protein expression of BL21 strain of Escherichia coli (E. coli) to be used instead of antibody. Methods: At first, complementary DNA (cDNA) construct encoding IL-5 was designed, and was ordered to be produced in pET28a vector. Expression vector was transformed into competent E. coli Bl21 (DE3) origami. Then, protein expression was optimized by altering temperature, incubation time, and the amount of isopropyl βd-1-thiogalactopyranoside (IPTG). Protein expression was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot in different levels of the test. Findings: The optimum conditions for protein expression were gained when the density of bacteria at the OD600 reached to 0.6 to 0.8, and culturing was done at 29 °C for 18 hours and 150 rpm, andinduction with 1mM IPTG. There was a 13-kDa protein band on SDS-PAGE and western blot that confirmed the expression of IL-5 protein. Conclusion: This protein can be used for producing aptamers against IL-5 and enzyme-linked immunosorbent assay (ELISA) kit for measuring IL-5. In all these process, there is no need to perfect folding of the protein. Therefore, the expression can be done in prokaryotic system, as it has high efficiency. © 2020 Isfahan University of Medical Sciences(IUMS). All rights reserved

Association of T/A polymorphism in miR-1302 binding site in CGA gene with male infertility in Isfahan population

Infertility occurs in 10-15 of couples worldwide and close to half of it is caused by male factors. One of the genes that can affect male infertility is CGA. Polymorphisms in CGA gene may affect gene expression, therefore affecting male infertility by disrupting the regulation of this gene. One of the polymorphisms is the substitution of T with A in the miR-1302 binding site in the 3' untranslated region of the CGA gene. In this study, we explored this polymorphism in Isfahan population. In this case-control study, by the use of Tetra primer-ARMS-PCR technique, rs6631 has been investigated in 224 infertile men and 196 controls. Infertile men were recruited from Isfahan Fertility and Infertility Center. Analysis of genotype and allele frequencies indicated that the differences between case and control populations were significant for rs6631 because P = 0.00 which is above the threshold. We found a significant relationship between this polymorphism and male infertility. This study which performed for the first time in Iran suggests that polymorphism in CGA gene can affect male infertility. Also, this polymorphism has high heterozygosity, so it can be used for further studies in different populations