Cloning and expression of EBI3 and p28 subunits of human interleukin 27 in E. coli

Interleukin [IL]-27 is a heterodimeric cytokine belonging to IL-12 and IL-23 families, secreted by antigen presenting cells [APCs]. The IL-27 is composed of 2 subunits: Epstein-Barr virus induced gene 3 [EBI3] and p28. IL-27 is an anti-inflammatory cytokine which has an inhibitory effect on Th17 population and suppress the IL-17 expression. It is suggested that IL-27 could be a potent drug candidate for treating auto immune diseases. The EBI3 and p28 subunits of human interleukin 27 were constructed into plasmid vectors; they sub-cloned into pETDuet-1 expression vector in restriction sites of BamHI, SacI and NotI. Subsequently the induction was carried out by 1mM IPTG and the recombinant proteins were confirmed by SDS-PAGE and Western Blot analysis using anti His-tag antibody. The EBI3 and p28 subunits of human interleukin 27were cloned into plasmid vectors. The 28 and 25kDa protein bands were observed on SDS-PAGE and finally confirmed by Western blot technique. In this research p28 and EBI3 proteins were produced successfully

New single nucleotide variation in the promotor region of androgen receptor [AR] gene in hypospadic patients

Hypospadias is one of the most common congenital abnormalities in the male which is characterized by altered development of urethra, foreskin and ventral surface of the penis. Androgen receptor gene plays a critical role in the development of the male genital system by mediating the androgens effects. In present study, we looked for new variations in androgen receptor promotor and screened its exon 1 for five single nucleotide polymorphisms [SNP] in healthy and hypospadias Iranian men. In our study, at first DNA was extracted from patients [n=100] and controls [n=100] blood samples. Desired fragments of promoter and exon 1 were amplified using polymerase chain reaction. The promoter region was sequenced for the new variation and exone 1 screened for five SNPs [rs139767835, rs78686797, rs62636528, rs62636529, rs145326748] using restriction fragment length polymorphism technique. The results showed a new single nucleotide variation [CT] at -480 of two patients' promoter region [2%]. None of the mentioned SNPs were detected in patients and controls groups [0%].This finding indicates that new single nucleotide polymorphism in androgen receptor promoter may have role in etiology of hypospadias and development of this anomaly