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Introduction: Background: Neff regulatory protein as well as structural proteins such as MPER and V3 of HIV-1 is important targets in vaccine studies. Therefore, in the current study, the Nef-MPER-V3 fusion protein was cloned and expressed in prokaryotic expression system that will be evaluated as a candidate for future vaccine studies. Materials and Methods: The Nef-MPER-V3 sequence was synthesized and inserted into pET-28a(+) vector using Not I/HindIII enzymes. Then, the recombinant construct was expressed in BL21 (DE3) strain of E.coli. For optimization of protein expression, several factors such as time of induction, IPTG concentration and optical density (OD) were evaluated. The recombinant protein was purified on a Ni-NTA agarose column and analyzed by 12% SDS-PAGE. Finally, the fusion protein was confirmed by western blotting. Results: The target gene was successfully cloned into the recombinant vector. The best condition for the expression such as; 2 m IPTG, OD600 = 0.7 and growth 5 hours post-induction were determined and the recombinant protein was purified by affinity chromatography in denaturing condition with urea at a concentration of about 300 μg/ ml. The purified fusion protein migrated as a clear band of around 35 kDa in SDS-PAGE and was detectable using anti-Neff antibody in western blotting. Conclusion: Our results showed that the Nef-MPER-V3 protein was successfully expressed in prokaryotic system and purified protein may provide the antigen for future experiments for immunogenicity evaluation in mice is currently undertaken. [ABSTRACT FROM AUTHOR]

Background and Objectives CD40L is a membrane protein and a member of the tumor necrosis factor super family (TNFSF) that plays an important role in transferring cell signaling in innate and adaptive immunity. Since this protein plays its role through clustering the receptors on the target cell surface, it seems that the multimeric form of this molecule can have a stronger effect than the natural trimeric form. So in this study we describe cloning and expression of dodecameric soluble CD40L through self multimerizing protein, surfactant protein D (SP-D), in the HEK293 cell line. Materials and Methods In this experimental study, the SPD-CD40L was designed in silico and cloned in pcDNA3.1(+) plasmid. HEK293 cells were transfected and used as the expression host cells. The expression of SPD-CD40L was determined at the RNA level by RT-PCR method. After the purification of the recombinant proteins by affinity chromatography, the molecular weight of the recombinant protein was determined by SDS-PAGE. To evaluate the specificity of protein, ELISA and Dot Blot were used. Results The results indicated that HEK293 cells were transfected and recombinant protein was expressed 24 and 48 hours after Transfection. Moreover, the results of ELISA and Dot Blot methods represented the specificity of recombinant SPD-CD40L chimeric protein. Conclusions Chimeric protein SPD-CD40L was expressed successfully by pcDNA3.1(+) in eukaryotic HEK293 cell line and the molecular weight of 4-trimeric (dodecameric) protein was shown to be consistent with the expected amount. [ABSTRACT FROM AUTHOR]

Background: SLC26A4 gene mutations are the second identifiable genetic cause of autosomal recessive non-syndromic hearing loss (ARNSHL) after GJB2 mutations. In this study, the identity and informativeness of D7S2459 marker in SLC26A4 gene region was examined in five ethnic groups of the Iranian population. Materials and Methods: The study was accomplished by genotyping the locus in 165 individuals of five different ethnic groups including Fars, Azari, Torkaman, Gilak and Arab using polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and fluorescent capillary electrophoresis. In this study, results were analyzed by GeneMarker HID Human STR Identity software, GenePop program and Microsatellite Tools software. Results: Analysis of the allelic frequency revealed the presence of 8 alleles of D7S2459 marker in the Iranian population. Among all, allele 148bp at the D7S2459 locus was the most frequent with 31% frequency. The most heterozygosity observed in Azari ethnic by 84.8%. Finally, analysis of PIC value revealed that D7S2459 marker could be considered as a highly informative marker in each ethnic of the Iranian population (PIC value above 0.7). Conclusion: Our data suggested that D7S2459 could be introduced as a highly informative marker in molecular diagnosis of SLC26A4 based ARNSHL by Linkage analysis. [ABSTRACT FROM AUTHOR]