arash arashkia
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ObjectiveCD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cellmalignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developinga bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTaggedanti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells.Materials and MethodsIn this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vectorand expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the elutedprotein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed inE. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher andSpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nbwas tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometryResultsSpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio ofSpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flowcytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is nobinding to the CD22- K562 control cells.ConclusionThe novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficialexpression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs).Keywords: CD22, Molecular Imaging, Single-Domain Antibody, Spycatcher, Spytag
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Background and Objectives
Although several studies have been achieved on the frequency of the HPV types among wom- en with cervical cancer in Iran, HPV-positive samples were in some cases directed to specific-primer genotyping of HPV 16 and 18. Therefore, the other HPV types are underestimated. Several studies have also reported a greater prevalence of HPV 16 in cervical cancer in Iran than in the world. To clarify these subjects, the distribution of HPV types in women referred for colposcopy in Tehran was investigated.
Materials and MethodsIn this cross-sectional study, a total of 148 cervical samples from women with normal, atypical squamous cells of undetermined significance, cervical intraepithelial neoplasia I-III, and invasive cervical cancer histopa- thology were included. HPV was detected by PCR assay and all HPV-positive specimens were subjected to direct nucleotide sequencing.
ResultsOur results demonstrated that the total prevalence of HPV was 92.5%. The five most common HPV types were HPV 16 (49.3%), 18 (14.8%), 6 (7.4%), 31 (4.1%), and 11 (2.7%). About the histopathological stage, HPV 16 and 18 were dominant in all studied groups. In cervical cancer, HPV 16 and 18 were detected in 60% and 20% of cases, respectively.
ConclusionHPV 16 and 18 were the most common in cervical cancer in Iran.
Keywords: Human Papillomavirus, Uterine Cervical Neoplasms, Cervical Intraepithelial Neoplasia, Atypical Squamous Cellsof The Cervix, Human Papilloma Virus Vaccines -
Background
Colorectal Cancer (CRC) represents a significant global health challenge, and its progression, resistance to therapy, and metastasis are strongly influenced by the tumor microenvironment, including factors like hypoxia. This study explores the impact of High Mobility Group Box 1 (HMGB1) overexpression on CRC cell migration, while identifying potential genes associated with this process.
MethodsTo explore this, we developed oncolytic virotherapy, resulting in HSVHMGB1, an oncolytic Herpes simplex virus that expresses HMGB1. HMGB1 is known its role in cancer progression, particularly in the context of cancer cell migration.
ResultsContrary to expectations, our scratch assays indicated that HSV-HMGB1 did not significantly induce migration in CRC cells, suggesting that HMGB1 might not directly contribute to this process. Employing microarray analysis, we investigated gene expression changes linked to CRC cell migration, leading to construction of a Protein- Protein Interaction (PPI) network. This network revealed the presence of hub proteins, including as NDRG1, LGALS1, and ANGPTL4, which are recognized for their roles in cancer cell migration. The differential expression of these genes under hypoxic conditions was further validated using quantitative RT-PCR, aligning with the findings from our microarray data.
ConclusionOur findings emphasize the complex regulation of CRC cell migration, and provides valuable insights into potential molecular mechanisms and pathways. These findings have implications for further research into cancer progression and the development of therapeutic strategies.
Keywords: Colorectal neoplasms, Galectin 1, HMGB1 protein, Oncolytic virotherapy, Simplevirus, Tumor microenvironment -
Background
The E6 oncoprotein of HPV plays a crucial role in promoting cell proliferation and inhibiting apoptosis, leading to tumor growth. Non-viral vectors such as nona-arginine (R9) peptides have shown to be potential as carriers for therapeutic molecules. This study aimed to investigate the efficacy of nona-arginine in delivering E6 shRNA and suppressing the E6 gene of HeLa cells in vitro.
MethodsHeLa cells carrying E6 gene were treated with a complex of nona-arginine and E6 shRNA. The complex was evaluated using gel retardation assay and FESEM microscopy. The optimal N/P ratio for R9 peptide to transfect HeLa cells with luciferase gene was determined. Relative real-time PCR was used to evaluate the efficiency of mRNA suppression efficiency for E6 shRNA, while the effect of E6 shRNA on cell viability was measured using an MTT assay.
ResultsThe results indicated that R9 efficiently binds to shRNA and effectively transfects E6 shRNA complexes at N/P ratios greater than 30. Transfection with R9 and PEI complexes resulted in a significant toxicity compared to the scrambled plasmid, indicating selective toxicity for HeLa cells. Real-time PCR confirmed the reduction of E6 mRNA expression levels in the cells transfected with anti-E6 shRNA.
ConclusionThe study suggests that R9 is a promising non-viral gene carrier for transfecting E6 shRNA in vitro, with significant transfection efficiency and minimal toxicity.
Keywords: Cell-penetrating peptides, E6 oncogene, Human papillomavirus 18, RNA interference -
Background
Hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. This study aimed to computationally identify hypoxia-related messenger RNA (mRNA) signatures in nine hypoxic-conditioned cancer cell lines and investigate their role during hypoxia.
MethodsNine RNA sequencing (RNA-Seq) expression data sets were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in each cancer cell line. Then 23 common DEGs were selected by comparing the gene lists across the nine cancer cell lines. Reverse transcription-quantitative PCR (qRT-PCR) was performed to validate the identified DEGs.
ResultsBy comparing the data sets, GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, PDK1, KDM3A, UBC, and P4HA1 were identified as hub genes. In addition, miR-335-5p, miR-122-5p, miR-6807-5p, miR-1915-3p, miR-6764-5p, miR-92-3p, miR-23b-3p, miR-615-3p, miR-124-3p, miR-484, and miR-455-3p were determined as common micro RNAs. Four DEGs were selected for mRNA expression validation in cancer cells under normoxic and hypoxic conditions with qRT-PCR. The results also showed that the expression levels determined by qRT-PCR were consistent with RNA-Seq data.
ConclusionThe identified protein-protein interaction network of common DEGs could serve as potential hypoxia biomarkers and might be helpful for improving therapeutic strategies.
Keywords: Hypoxia, MicroRNA, RNAseq -
Background
Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for Semliki Forest virus (SFV)-PD self-amplifying mRNA (SAM) preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of virus replicon particle (VRP)-packaged SAM for vaccine studies.
MethodspSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into human embryonic kidney 293 cells (HEK-293) and baby hamster kidney cell line 21 (BHK-21) cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by reverse transcription quantitative real-time PCR (RT-qPCR).
ResultsIn vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR.
ConclusionUsing optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.
Keywords: mRNA vaccines, Semliki Forest virus, Vaccines -
Introduction
Cervical cancer is one of main causes of cancer death in women, especially in developing countries. Therefore, a low-cost broad-spectrum preventive vaccine is immediately needed. The RG-1 epitope of L2 protein is a major cross-neutralizing epitope but has low immunogenicity. This defect can be overcome by using built-in adjuvants such as TLR agonists and bacterial toxoid epitopes. To address this issue, we designed an epitope-based vaccine against HPV16 using immunoinformatic tools.
MethodsThe HPV16 RG-1 epitope was linked to built-in adjuvants including the D1 domain of flagellin and RS09 epitope as TLR agonists, and a tetanus toxoid epitope for induction of immune responses. Using immunoinformatic tools, the immunological characteristics of the construct were evaluated. In the first step, MHC-I and II binding, CD4+ T cell immunogenicity prediction, and in the second step, immunogenicity simulation of the construct were investigated.
ResultsMHC-I and II predicted epitopes showed a high potentiality to bind to mice and human MHC alleles. The results of the binding of the RG-1 epitope to MHC-I and MHC-II showed that RG-1 could induce humoral and cellular immune response while fused to three built-in adjuvants. Also, the CD4+ immunogenicity assessment results predicted that several epitopes in the designed construct, including epitopes of D1 domain and tetanus toxoid P2 epitope, behaved as potentially strong Th inducers. The immunogenicity simulation results showed that the construct could potentially provide sufficient antigen, and induce suitable humoral and cellular immune responses.
ConclusionThe development of new vaccine strategies has been the focus of several studies. The results showed that the designed construct can potentially provide an effective model for developing a preventive vaccine candidate against a variety of HPV genotypes.
Keywords: HPV16 RG-1, Built-in adjuvants, TLR agonists, Immunoinformatic, immunogenicity simulation -
Measles virus, negative-strand RNA viruses, has been known as an ideal candidate in oncolytic virotherapy. Recombinant measles virus can encode genes of interests for reaching several aims. Replication efficiency of oncolytic virus in tumoral cells is a key parameter in efficient tumor eradication. Products of P gene (P/V/C) support measle virus to circumvent IFN 1 as the main response of innate immune system against viruses. But vaccine strains used in oncolytic therapy studies comprise several mutations in their P gene sequences. These mutations affect replication efficacy which cause attenuation of measles strains applicable in vaccination. So, arming vaccine strains with the wild type P gene is helpful to reach high virus titer. Here at this study, we have expanded a protocol with details for engineering and efficient recovery of measles virus for different aims.
Keywords: Measles Virus, Reverse Genetics, Viral Rescue, Oncolytic Virotherapy -
Background
Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe E. coli expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for ex vivo stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells.
MethodsA bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the HindIII and BamHI site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the E. coli (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole.
ResultsAnalyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein.
ConclusionsResults showed efficient production of the bsAb in E. coli for future large-scale purification.
Keywords: Bispecific Antibody, Escherichia Coli, Immunotherapy, Natural Killer Cell (NK Cell), Newcastle Disease Virus (NDV) -
Acute gastroenteritis is one of the most important causes of death in children in developing countries which cause by different enteropathogens, including bacteria, viruses, and parasites. Among these, most of the acute gastroenteritis in children are caused by viral infections mainly by rotavirus and norovirus. This study aimed to study the epidemiological and clinical status of acute gastroenteritis resulting from rotavirus and norovirus in children between June 2015 and June 2016 in Iran. A total of 211 stool specimens were collected from Ali Asghar Children's Hospital and Bahrami Children's Hospital in Tehran, from June 2015 to June 2016. The samples were screened by commercial enzyme immunoassay (EIA) Ridascreen kit and real time RT-PCR to detect rotavirus and norovirus genogroups I and II, respectively. The information on demographic and clinical manifestations was collected, and data analyzed using IBM SPSS statistics version 22. Overall, the detection rate of rotavirus was 25.6 %, and for norovirus infection, it was 17.5%. All norovirus positive specimens belonged to genogroup II. Higher rates of rotavirus infections were observed in children from 7 to 24 months, and higher rates of norovirus infections were detected in children from 1 to 12 months. Clinical symptoms were not different between rotavirus and norovirus case-patients. The present study not only highlights the importance of rotavirus and norovirus infections in Iran but also verifies the relevance of norovirus as the cause of severe gastroenteritis in children.
Keywords: Rotavirus, Norovirus, Acute gastroenteritis, Clinical characteristics -
Background
Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct.
MethodsIn this study, a CAR was constructed based on a nanobody against PSMA (NBPII-CAR). At first, Jurkat cells were electroporated with NBPII-CAR, and then flow cytometry was performed for NBPII-CAR expression. For functional analysis, CAR T cells were co-cultured with prostate cancer cells and analyzed for IL-2 secretion, CD25 expression, and cell proliferation.
ResultsFlow cytometry results confirmed the expression of NBPII-CAR on the transfected Jurkat cells. Our data showed the specificity of engineered Jurkat cells against prostate cancer cells by not only increasing the IL-2 cytokine (about 370 pg/ml) but also expressing the T-cell activation marker CD25 (about 30%). In addition, proliferation of engineered Jurkat cells increased nearly 60% when co-cultured with LNCaP (PSMA+), as compared with DU145 (PSMA-).
ConclusionHere, we describe the ability of nanobody-based CAR to recognize PSMA that leads to the activation of Jurkat cells. This construct might be used as a promising candidate for clinical applications in prostate cancer therapy.
Keywords: Chimeric antigen receptor, Immunotherapy, Prostate cancer, PSMA -
Background
Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SKg). Based on the variable region of SKβ, GAS-SKs (ska) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SKβ exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented.
MethodsTwo parental SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SKC1: αG88-βSTAB-γG88 and SKC2; αSTAB-βG88-γSTAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs were measured by colorimetric assay.
ResultsSDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SKs. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aβ was around six times, whereas for SKG88/SKC2 containing skcgβ, it was four times.
ConclusionAlthough SKβ has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.
Keywords: Plasminogen, Streptokinase, Thrombolytic therapy -
International Journal of Travel Medicine and Global Health, Volume:6 Issue: 1, Winter 2018, PP 7 -10
Rotaviruses are the most common cause of severe diarrhea in children under 5 years of age worldwide with a higher prevalence in developing countries. In accordance with the World Health Organization (WHO) recommendations for the global use of rotavirus vaccines, it is important to review trends of rotavirus epidemiology, distribution and diversity of rotavirus strains in the pre-vaccine period. In Iran, the average rotavirus positivity rate is 40.04% in all patients under 5 years of age hospitalized for acute gastroenteritis (AGE). Studies have shown a substantial increase in the rotavirus detection rate over time from 1986 to 2013. Moreover, there has been continued predominance of G (G1) and P (P[8]) genotypes, although the peak prevalence of G1 appeared to decline in 2007-2011 compared to 2001-2006. The data presented in this review, which suggests a change in the pattern of rotavirus genotypes in the Iranian population, further highlights the important role of continuous monitoring of rotavirus genotypes before starting any national rotavirus vaccination program.
Keywords: Rotavirus Group A, Epidemiology, Genotype, Iran -
پروتئین(High mobility group box 1) HMGB1، پروتئین داخل سلولی است که به هسته انتقال می یابد و بیان ژن ها را تنظیم می کند. پروتئین HMGB1 به عنوان یک پروتئین چند عملکردی نقش خود را از طریق نوترکیبی، تنظیم رونویسی و التهاب ایفا می کند. پروتئین HMGB1 به کیناز وابسته به سایکلین مانند CDK2 متصل می شود که رونویسی ژن های مرتبط با پیشرفت چرخه سلولی را کنترل می کند. به علاوه HMGB1 به عنوان سایتوکاین جدید نقش مهمی در التهاب و بیماری هایی نظیر آرتریت دارد. به تازگی، نقش پروتئین HMGB1 به عنوان ادجوانت (یاور) و محرک هر دو پاسخ ایمنی سلولی و همورال (سرمی) در عفونت های ویروسی نظیر HIV-1 و آنفلوانزا به اثبات رسیده است. برخی مطالعه ها نشان دادند که Hp91، پپتید مشتق شده از پروتئین HMGB1، به عنوان محرک موثر و قوی برای بلوغ دندرتیک سل (DC) عمل کرده و توانسته است پاسخ های ایمنی سلولی و همورال را تحت شرایط درون تنی تقویت کند. یافته های اخیر ما نیز نشان داد که پپتید Hp91 و طول کامل ژن HMGB1 توانسته اند به عنوان ادجوانت موثر به منظور بهبود کارآیی واکسن های درمانی پروتئینی و DNA یی در مقابل عفونت ویروس پاپیلومای انسانی به ترتیب عمل کنند. در این مطالعه، خلاصه ای از ساختار و عملکردهای HMGB1 در بیولوژی مولکولی و پزشکی شرح داده می شود.کلید واژگان: ادجوانت اندوژنوس, HMGB1, Hp91, فعالیت بیولوژیکیHMGB1 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.Keywords: Endogenous adjuvant, HMGB1, Hp91, Biological activity
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زمینه و هدفویروس پاپیلوم انسانی(HPV)نقش مهمی در برخی از بدخیمی های انسانی ایفاء کرده و منجر به تغییر سطوح بیان نرمال ریز آر ان ای های سلولی می شود. در این مقاله اثرات چنین تغییراتی بر روی نمونه های تومور سرطانی سنگفرشی سر و گردن در سطح پروفیل ژنی بررسی شد.روش هادر این مطالعه توصیفی- تحلیلی پروفیل بیان ژنی 36 نمونه تومور در دو گروه با و بدون ویروس مقایسه شدند. ژن های با بیان متفاوت در دو گروه از لحاظ توانایی تفکیک نمونه ها و نیز همپوشانی با دسته بندی های هسته شناسی ژنی سنجیده شدند. به علاوه، اثر اهداف سلولی تایید شده 11 ریز آر ان ای های سلولی گزارش شده بر روی پروفیل بیان ژنی نمونه های تومور با استفاده از آنالیز خوشه بندی سلسله مراتبی و آنالیز غنی سازی مجموعه ژنی بررسی گردید.نتایجبر خلاف آزمون های غیر نظارتی، ژن های استخراج شده با بیان متفاوت، مشتمل بر 47 و 7 ژن واحد به ترتیب با بیان افزایش یا کاهش یافته، به خوبی دو گروه تومور را در آنالیز خوشه بندی سلسله مراتبی تفکیک کرده (0/0001=P). این ژن ها و عمدتا در تنظیم چرخه سلولی نقش داشتد (0/05q-value≤). اهداف ریز آر ان ای های سلولی با بیان افزایش یافته در گروه توموری با ویروس غنی تر یافت شدند (0/05q-value≤). در بین ریز آر ان ای های سلولی بررسی شده، hsa-miR-155-5p و hsa-miR-221-3p به صورت معنی داری پروفیل بیان ژنی نمونه های تومور را تغییر دادند (0/05q-value≤).نتیجه گیریویروس پاپیلوم انسانی با تغییر سطوح بیان ریز آر ان ای های سلولی می تواند بر روی پروفیل بیان ژنی سرطان سلول های سنگفرشی سر و گردن اثر گذارد. تایید نتایج این مطالعه با استفاده از روش های آزمایشگاهی پیشنهاد می شود.کلید واژگان: ویروس پاپیلوم انسانی, سرطان سلول های سنگفرشی سر و گردن, ریز آر ان ای, پروفیل بیان ژنیBackground And AimHuman 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.Methodsin 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.ResultsUnlike 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).Conclusionderegulating 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.Keywords: Human Papilloma Virus, Squamous cell carcinoma of the head, neck, microRNA, Gene Expression Profiling
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Current licensed and commercially available prophylactic human papillomavirus (HPV) vaccines (Cervarix and quadrivalent/nine valents Gardasil) are based on major capsid protein L1 virus-like particles (VLPs) production which are expensive and type specific. Minor capsid L2-RG1 linear epitope (17-36) is a known candidate for induction of cross-neutralizing antibodies to develop low-cost pan-HPV vaccines. Herein, we report construction and expression of a three tandem repeats of L2-RG1 derived from HPV16 and 18 fused with the same head to tail pattern (HPV16:17-36×3 HPV18:17-36×3; hereafter termed dual-type fusion L2 peptide) in E. coli and provide the results of its immunogenicity in mice. SDS-PAGE and western blot analyses indicated proper expression of the peptide that could be further purified by Ni-NTA affinity chromatography via the located C-terminal 6xHis-tag. Mice immunized by formulation of the purified peptide and Freund adjuvant raised neutralizing antibodies which showed proper cross reactivity to HPV L2 (11-200) of types: 18, 16, 31 and 45 (which totally are responsible for 90% of cervical cancers) and efficiently neutralized HPV18/16 pseudoviruses in vitro. Our results imply the possibility of development of a simple, low-cost preventive HPV vaccine based on this dual-type fusion L2 peptide in bacterial expression system with broad spectrum.Keywords: Human papilloma virus, Cross neutralization, Pseudovirus, L2 peptide
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بیان موقت، یکی از روش های موثر برای تایید کارایی پیشبرها در بیان ژن هدف در گیاهان است. در پژوهش حاضر، بیان آنتی ژن سطحی هپاتیت ب (HBsAg) در کنترل پیشبر دایمی CaMV35S و همچنین پیشبرهای اختصاصی بافت میوه گیاه گوجه فرنگی (E8 و 2A11) بررسی شد. بدین منظور از روش آگرواینفیلتریشن در بافت های برگی گیاه توتون و گوجه فرنگی و همچنین میوه گیاه گوجه فرنگی استفاده شد. نتایج حاصل نشان داد که دو پیشبر اختصاصی میوه، همانند پیشبر قدرتمند و دایمی CaMv35S، بیان زیاد آنتی ژن را در میوه های گیاه گوجه فرنگی باعث می شوند. اگرچه آنتی ژن HBsAg در بافت های برگی هم بیان شد، میزان آن نسبت به بافت میوه کمتر بود. شاید تولید اتیلن بر اثر زخمی شدن قطعات برگی، القای پیشبرهای اختصاصی میوه را باعث شده که خود بیان آنتی ژن مربوط را سبب شده است. نتایج پژوهش حاضر نشان می دهد که از پیشبرهای اختصاصی میوه گوجه فرنگی (E8 و 2A11) می توان به طور موثری برای بیان دایمی آنتی ژن HBsAg در گیاه گوجه فرنگی تراریخت استفاده کرد.کلید واژگان: بیان موقت, پیشبر اختصاصی, گوجه فرنگی, هپاتیت ب, _ HbsAgTransient 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.Keywords: HBsAg, Hepatitis B, Specific promoters, Tomato, Transient Expression
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مجله دانشکده پزشکی دانشگاه علوم پزشکی تهران، سال هفتاد و سوم شماره 9 (پیاپی 177، آذر 1394)، صص 624 -631زمینه و هدفویروس پاپیلومای انسانی (HPV) یکی از شایع ترین ویروس هایی است که در ایجاد سرطان دهانه رحم نقش دارد و پژوهش ها نشان داده است که در بیشتر موارد سرطان دهانه رحم، پروتئین های E6 و E7 ویروس پاپیلوما وجود دارد. هدف از مطالعه کنونی بهینه سازی بیان سازه پلی توپیک حاوی ژن های E6 و E7 ژنوتیپ های مختلف ویروس پاپیلومای انسانی به عنوان کاندیدای واکسن بود.روش بررسیاین مطالعه تجربی از مهر 1392 تا شهریور 1393 در بخش ویروس شناسی انستیتوپاستور ایران انجام شد. ابتدا نواحی ایمنی زای پروتئین های E6 و E7 در ژنوتیپ های مختلف پاپیلوما ویروس انسانی (HPV16، 18، 31، 45) به صورت بیوانفورماتیک مورد بررسی قرار گرفتند. سازه بیانی سنتتیک کدکننده پروتئین های E6 و E7 در اشرشیاکلی ترانسفورم شده و سپس بیان پروتئین مربوطه انجام گرفت و به منظور بهینه سازی بیان پروتئین نوترکیب مورد نظر، دانسیته سلولی، مدت زمان انکوباسیون، دمای رشد، غلظت های مختلف isopropyl-beta-D-thiogalactopyranoside (IPTG) و محیط های کشت مختلف مورد استفاده قرار گرفت.یافته هاپس از بهینه سازی بیان، بهترین بیان پروتئین نوترکیب مورد نظر در محیط کشتSuper Optimal Broth (SOB) (Merck، Germany) به دست آمد. در این محیط کشت، بیش ترین میزان بیان در 8/0(Optical density) OD600=، غلظت mM 1/0 از IPTG، زمان انکوباسیون یک ساعت در دمای °C 37 در میزان بیانی BL21 (A1) اشرشیاکلی به دست آمد.نتیجه گیرینتایج حاصل از این مطالعه نشان داد که بیان پروتئین های E6 و E7 ژنوتیپ های مختلف ویروس پاپیلومای انسانی قابل انجام بوده و در نهایت می توان از آن به عنوان یک کاندیدای واکسن علیه ویروس استفاده نمود.
کلید واژگان: ویروس پاپیلومای انسانی, پروتئین E6 و E7, واکسن های پلی توپیک, اشرشیاکلیBackgroundHuman 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.MethodsIn 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.ResultsIn 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.ConclusionThe 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.Keywords: human papilloma virus, E6, E7 proteins, polytopic vaccine, E.coli -
Background
The key enzyme in biotin‑(strept) avidin systems, Escherichia coli BirA biotin ligase, is currently obtained by overexpression of the long protein‑tagged versions of the gene to prevent its toxic effect in E. coli. Herein we describe a rather simple and efficient system for expression of E. coli BirA without the application of long‑tag proteins.
Materials and MethodsThe coding sequence of BirA gene was isolated by polymerase chain reaction using DNA extract of E. coli‑DH5α as template. BirA amplicon harboring a GS‑linker at its C‑terminal was cloned into NdeI‑XhoI sites of pET24a(+) vector under control of T7 promoter and upstream of the vector‑derived 6xHis‑tag. pET24‑BirA transformed BL21‑cells were induced for protein expression by IPTG and analyzed by SDS‑PAGE and Western blotting. Protein expression yields were assessed by image analysis of the SDS‑PAGE scans using ImageJ software.
ResultAgarose gel electrophoresis indicated proper size of the BirA gene amplicon (963 bp) and accuracy of the recombinant pET24‑BirA construct. Sequence alignment analysis indicated identical sequence (100%) of our isolate with that of the standard E. coli‑K12 BirA gene sequence (accession number: NC_000913.3). SDS‑PAGE and Western blot results indicated specific expression of the 36.6 kDa protein corresponding to the BirA protein. Image analysis estimated a yield of 12% of total protein for the BirA expression.
ConclusionsBy application of pET24a(+) we achieved relatively high expression of BirA in E. coli without application of any long protein‑tags. Introduction of the present expression system may provide more readily available source of BirA enzyme for (strept) avidin–biotin applications and studies.
Keywords: Biotin–(strept) avidin, biotin ligase, BirA, E. coli, pET24a(+) -
زمینه و اهدافتاثیرات سرکوب کنندگی برخی از کلیدی ترین پروتئین های ویروس هپاتیت c بر سیستم ایمنی و جهش سریع ویروس، توجه را به واحدهای ایمونوژن کوچک و پایدار جلب نمود. لذا، واکسن های مولتی اپی توپ به منظور تمرکز پاسخ ایمنی بر این واحدها معرفی شدند. ولی، به دلیل پاسخ ایمنی نسبتا ضعیف علیه این واکسن ها، افزایش ایمنی زایی نیازمند تمهیدات بیشتری است. این مطالعه با هدف ساخت پلاسمیدهای حامل اپی توپ های CTL ویروس هپاتیت C به روش مدل سازی ایمونوانفورماتیک و تعیین اولیه ایمنی زایی آنها انجام شد.روش بررسییک اپی توپ H-2Dd و دو اپی توپ HLA-A*0201 از نواحی E2، E1 و core در HCV انتخاب شد و با ابزارهای ایمونوانفورماتیک برای بهترین ترادف آنالیز شد. توالی مولتی اپی توپ به روش SOEing-PCR ساخته شد و در ناقل pcDNA3.1+ کلون گردید. توالی های ارتقاء دهنده PADRE، سیگنال رتیکولوم آندوپلاسمیک (ER signal sequence: ERss) و HBsAg به انتهای ''5 توالی مولتی اپی توپ متصل شد و بیان پلاسمیدها به صورت in-vitro به روش های RT-PCR، دات-بلات، وسترن-بلات و ایمونوفلورسانس بررسی شد. ارزیابی اولیه ایمنی زایی به صورت in-vivo با آزمایش ازدیاد حساسیت تاخیری (Delayed type hypersensitivity: DTH) انجام شد. نتایج با آزمون های Non parametric Mann-Whitney و ANOVA ارزیابی شدند.یافته هانتایج آزمایش های in-vitro، بیان پلاسمیدها را تائید کرد و ارزیابی های اولیه in-vivo، پردازش و عرضه اپی توپ H-2Dd را در موش BALB/c نشان داد. همچنین در حالیکه اثرات ارتقاء پاسخ برای ERss و PADRE نشان داده شدند، اتصال HBsAg تاثیری در افزایش پاسخ نداشت.نتیجه گیریاین مطالعه ارزش پیش بینی های ایمونوانفورماتیک و آزمایش DTH را، برای آنالیز اولیه واکسن-های داوطلب مولتی اپی توپی در موش های BALB/c، نشان داد. نتایج، تائید کافی برای بررسی بیشتر سازه های طراحی شده را در موش ترانس ژنیک (تراریخته) HLA-A2 فراهم می کند.
کلید واژگان: ایمونوانفورماتیک, تقویت کننده ایمنی, مولتی, اپی توپ, بیان یوکاریوتی, ویروس هپاتیت CBackground And ObjectivesHigh 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.Materials And MethodsOne 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.ResultsIn 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.ConclusionThis 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.Keywords: Hepatitis C Virus_Immunoinformatics_Multiepitope vaccine_In vivo expression_Immunostimulant
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