آرش آرش کیا

Interleukin 15 (IL-15) is a cytokine that, due to its physiological activity, can play a role in anti-cancer therapies. It shows positive effect on Natural Killer cells differentiation, proliferation, activation, and surveillance and also on surveillance of memory CD8+ T cells. However, the expression and purification yield of recombinant IL-15 is low. So, it worth improving the production conditions of this useful protein. Therefore in the present study, cloning and expression of human IL-15 are reported in E. coli, strain Rosetta (DE3) which is different from previous bacterial hosts BL21 strain. This strain (Rosetta DE3) has the potential to use codons rarely used by bacteria and consequently, the expressed protein can be more similar to the human protein. First, the human IL-15 coding sequence was synthetized, and then the sequence was cloned into pET28a plasmid. Confirming the accuracy of the final construct was done by colony PCR, restriction analysis (which was done using BamHI and XhoI restriction enzymes and the expected 391bp band was observed) and sequencing. Then, the recombinant construct was transformed into competent E. coli Rosetta (DE3) bacteria. Expression was done in OD600 of 0.6 in the presence of 1mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG). Protein characterization was done by SDS-PAGE and then by western blotting using a specific commercial anti-IL-15 antibody. The 15kDa band on the gel and blot, showed the presence of IL-15. Densitometry by Fiji software determined 37% production yield. It is expected a suitable function from this produced recombinant cytokine due to expression in E. coli Rosetta (DE3) in future studies.

 The chimeric domain-exchanged streptokinase (SKch) between two sk genes from groups G and A streptococci (SK2aG88 and SK2bALAB49, respectively) was constructed to evaluate the role of SK-domains (α, β, γ) in α2-antipalsmin-resistance variations of SK.

 In this experimental study, PCR-amplified genes of streptococci (skg, ska) were cloned into pET26b vector to produce pET26-SKG88 and pET26-SKALAB49. For domain exchange, the amplicon containing β and γ domains of SK2bALAB49 was replaced for that of the SK2aG88 within pET-SK2aG88 (pET26-SKch; α2aG88β2bALABγ2bALAB). All constructs were confirmed by restriction analyses/agarose-gel electrophoresis and DNA sequencing, transformed into E.coli Rosetta, and induced by IPTG for protein expression. Proteins were purified by Ni-NTA chromatography, quantified by Bradford method, and analyzed by SDS-PAGE/Western blotting assays. The α2-antipalsmin-resistance was measured by S2251 colorimetric assay for plasminogen activation.

 SDS-PAGE and western blotting results indicated the expression of proteins with the size of 47kD. At the highest concentration of α2-antiplasmin, SK2aG88 remained 80% active, whereas the SK2bALAB49 and SKch retained 55% of their activity.

 SKch shows similar activity reduction, indicating the minor role of the α domain compared to β and γ domains for α2-antipalsmin-resistance.

HMGB1 protein is an intracellular protein that translocated into the nucleus and regulates genes expression. HMGB1 plays multi-functional roles through recombination, transcriptional regulation, and inflammation. HMGB1 binds to cyclin-dependent kinase such as CDK2 that regulates the transcription of genes associated with the progression of cell cycle. In addition, HMGB1 as a novel cytokine plays major role in inflammation and arthritis. Recently, the role of HMGB1 protein as an adjuvant and stimulating both humoral and cellular immune responses has been proven in viral infections such as HIV-1 and influenza. Several studies indicated that Hp91, a HMGB1-derived peptide, stimulates efficiently DC maturation, and boosts the cellular and humoral immune responses in vivo. Our recent data also showed that the full length of HMGB1 gene, and Hp91 peptide could be an effective adjuvant in developing the therapeutic HPV DNA- and protein-based vaccines, respectively. In this review, the structure and functions of HMGB1 is described in molecular biology and medicine.

Human Papilloma Virus plays an important role in some of human malignancies and causes alterations in normal expression levels of cellular microRNAs. In this paper, we evaluated the effects of such changes on Head and Neck Squamous Cell Carcinoma tumor samples at gene expression profile level.
in this descriptive-analytical study, gene expression profiles of 36 tumor samples were compared in two groups: with or without virus. Differentially expressed genes among the two groups were judged in terms of their ability in segregating the tumor samples and also their overlap with Gene Ontology Biological Function categories. Furthermore, using hierarchical clustering analysis and Gene Set Enrichment Analysis methods, the effect of confirmed cellular targets of 11 reported cellular microRNAs on the gene expression profiles of our samples was assessed.
Unlike unsupervised methods, differentially expressed genes, including 47 and 7 unique induced and suppressed genes, respectively, discriminated perfectly the two sample sets in a hierarchical clustering analysis (P=0.0001). These genes were primarily engaged in regulation of cell cycle (FDR adjusted P≤0.05). Targets of induced cellular microRNAs were found enriched in virus-positive set (FDR adjusted P≤0.05). Among analyzed cellular miRNAs, hsa-miR-155-5p and hsa-miR-221-3p change the gene expression profile of tumor samples significantly (FDR adjusted P≤0.05).
deregulating expression levels of cellular microRNAs, HPV is capable of affecting the gene expression profiles of Head and Neck Squamous cell Carcinoma tumors. It is suggested to confirm the results of this study using experimental methods.

Transient gene expression experiments are especially useful to confirm the efficiency of promoters used for expression of foreign molecule in plants. In this study, the expression of the hepatitis B surface antigene (HBsAg) under the control of the CaMV35S and fruit tissue-specific (E8 and 2A11) promoters were studied in the leaf tissues of tobacco and tomato plants and tomato fruits using agroinfilteration technique. The results showed that fruit-specific promoters as the constutative CaMv35S promoter cause over-expression of the antigen in tomato plant fruits. However, HBsAg antigen was expressed in the leaf tissues, but its rate was lower than the fruits. Ethylene produced by the wounded leaf parts may induce fruit-specific promoter, which in turn is related antigen expression. The results show that the tomato fruit specific promoters (E8 and 2A11) can be used as for efficient expression of HBsAg antigen in transgenic tomato plants.

Human papilloma virus is a DNA virus from the papillomavirus family that is most prevalent in human cervical cancers and many studies showed the E6 and E7 proteins are present in the majority of cervical cancer cases. Development of universal HPV peptide-based vaccine with more serotypes coverage has considerable value. The aim of the study was to design a multi-epitope universal vaccine for major HPV based on E6 and E7 proteins and optimization the expression of polytopic construct contains E6 and E7 genes from different genotypes of human papilloma virus as a candid vaccine. In this experimental study that was carried out in Pasteur Institute of Iran, Virology Department from October 2013 to November 2014. In order to design the polytypic construct, we predicted the most probable immunogenic epitopes of E6 and E7 from common high risk HPV16, 18, 31, 45 along with high prevalent type 6 and 11 using bioinformatics methods. The synthetic pET28a expression vector harboring E6 and E7 protein was transformed into Escherichia coli hosts and its expression was analyzed by SDS-PAGE and western blotting. Finally, in order to expression optimization of recombinant protein, cell density, induction time, growth temperature, IPTG (Isopropyl β-D-1-thiogalactopyranoside) concentration and cultures media were studied. In the present study the recombinant fusion protein was expressed successfully and the highest expression of target protein was achieved in super broth medium containing 0.1% glucose and 0.2% L-arabinose. In Super broth medium, the optimum condition for recombinant protein expression was occurred at OD600 of 0.8, 0.1mM IPTG, one hour’s incubation time at 37 °C and BL21 (A1) host. The results of this study show that the optimum expression of E6 and E7 proteins from different genotypes of human papilloma virus can be performed. Moreover, by purification of recombinant protein and evaluation of its immunogenicity in mice, it can be used as a vaccine candidate against the human papilloma virus.

High mutation rate and immunosuppressive effect of some key proteins of hepatitis C virus (HCV) encouraged researchers to find conserved and small immunogenic units to induce immune responses. Thus epitope-based vaccines were introduced to find out any potential immunogens. However, the relatively weak immune responses against these vaccines require a creation of additional measures. This study aimed to construct plasmids carrying CTL epitope of hepatitis C virus using immunoinformatics modeling method and assessment of their preliminary immunogenicity. One H-2Dd and two HLA-A*0201-restricted CD8+ T-cell epitopes from E2, E1 and core regions of HCV were selected and immunoinformatically analyzed for optimum sequentiality. The multiepitope sequence was constructed by SOEing PCR and cloned in pcDNA3.1+ vector. PADRE, endoplasmic reticulum signal sequence (ERss) and hepatitis B surface antigen (HBsAg) immuno-enhancer sequences were fused to the 5′ end of multiepitope constructs. In vitro, expression of each plasmid was analyzed by RT-PCR, dot-blot, Western-blot and immunofluorescence techniques. Preliminary in vivo immunogenicity of the construct was assessed by delayed-type hypersensitivity (DTH) response in BALB/c mice. In vitro, analyses confirmed the expression of plasmids. Preliminary, in vivo assessments indicated the processing and presentation of the H-2Dd epitope in BALB/c mice. Moreover, although the immunostimulation effects of ERss and PADRE were shown, fusion of HBsAg did not enhance the DTH response. This study shows the value of immunoinformatics prediction of hepatitis C virus epitopes as a multiepitope vaccine candidate and DTH assay for preliminary analysis using BALB/c mice. Data obtained, provide enough support for further evaluation of the designed constructs in HLA-A2 transgenic mice.