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Purpose: The antigen binding fragments of camelids heavy chain antibodies are comprised in one single domain (VHH). The crystal structure of an isolated VHH indicated that it is a problate particle of 2.5 nm in diameter and ∼ 4 nm high, and has been referred to as a nanobody. The very close similarity of these molecules to human VHs illustrates the potential application of these novel products as an immunodiagnostic immunotherapeutic reagent. One of the key components in production, characterization and application of nanobodies in detection is the anti-nanobodiy HRP conjugate. Materials and Methods: Here, we report high expression and purification of some nanobodies against tumor markers. The nanobodies genes were sub-cloned into a pSJF9 vector to over-express the protein coupled with fusion tags in E. coli TG1. The expressed nanobodies were purified by immobilized metal affinity chromatography (IMAC). Described here is the preparation, purification and characterization of anti-nanobody antibody HRP conjugate for use in the various nanobody detection systems. Results: Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the purified nanobodies. Because of application of several biochemical modifications, the produced anti-nanobodies HRP conjugate have efficient sensitivity and specificity. Conclusion: By setting the temperature, time and inducer reagent the nanobodies were produced in optimum yield. We concluded that the HRP conjugated anti-nanobody can detect nanobodies in various detection procedures with great sensitivity and accuracy.

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Objective: The greatest challenge in cancer gene therapy is to achieve the high specificity and efficiency in targeting of cancer cells. Because the goal of cancer gene therapy is to eradicate cancer cells, many therapeutic genes could be detrimental if unintentionally expressed in normal cells. Using promoter of the genes which are expressed specifically in cancer cells or have much more expression in cancer cells than normal cells, is very noticeable tool in cancer gene therapy (CGT). In this study we were searching for cancer specific promoter which could highly express therapeutic gene. Materials and methods: In order to apply a cancer specific promoter for creating a CGT construct, a promoter which have 34% similarity to Survivin core promoter was amplified from human genome by using Nested-PCR. Survivin is a member of anti-apoptotic gene and its over-expression was observed in up to 70% of breast cancers. This gene fragment contains two transcriptional binding sites which were similar to Survivin promoter according to the evaluation of Promoter Scan, EPD, Transfac, Compel and TRRD program. These binding sites were recognized by STAT1 and E2F transcription factors. This promoter was cloned into pCDNA3.1/Hygro+ plasmid in along with hypoxia and estrogen modules and pro-apoptotic gene tBid. Results: Semi-quantitative RT-PCR results of transfected cancer cells showed that this gene fragment (Survivin like promoter) have relatively same potential as CMV promoter to direct tBid gene expression. Conclusion: Utilization of chimeric promoter containing Survivin like promoter could be a promising tool in killing cancer cells naturally by inducing apoptosis. This construct is highly effective in transcriptional targeting of tBid in comparison to control construct.

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Objective: Today, AIDS is considered as a global problem and many efforts to generate an effective vaccine against this disease have been made, but remain inconclusive. DNA vaccines are a member of the new generation of vaccines that can efficiently stimulate the immune system. However, recent findings indicate low immunogenicity for these vaccines and it is believed that these types of vaccines require strategies that could infer more immunogenicity. The employment of adjuvants could be considered as one of the most important methods involved. In this study, a DNA vaccine candidate for HIV P24-Nef is constructed and then using genetic adjuvants IL-15 and GM-CSF, cellular immune responses have been studied. Materials and Methods: In this study the gene structure of HIV P24-Nef in eukaryotic expression vector was constructed and expression vectors of IL-15 and GM-CSF were used as adjuvants. After inoculation of the candidate vaccine to BALB/c mice, cytokine patterns, lymphocytes proliferation and cytotoxicity were analyzed. Results: Our findings indicate that candidate vaccine significantly stimulated cellular immune responses. The usage of IL-15 and GM-CSF as DNA adjuvants together and separately with candidate vaccine has strengthened cellular immune responses significantly. Co-administration of DNA adjuvants significantly increased cellular immune responses when the ratio of the vaccine dose was more than the adjuvants. Conclusion: The sequences that we selected as candidate vaccine demonstrated good immunogenicity in mouse model and co-administration of IL-15 and GM-CSF DNA adjuvants increased cellular immune response to DNA vaccine construct.

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Objective: Several vaccines against HIV have been investigated but none has been approved as an effective HIV vaccine. An approach that could induce stronger immune response against the pathogen is utilizing a multi-epitopic vaccine. This strategy was used in the design of several vaccines and resulted in improved immune responses. Materials and Methods: In this study a multi-epitopic fusion peptide including parts of HIV-1 Nef and P24 as a vaccine candidate was injected into mice and immune humoral responses measured with total antibody and IgG sub-classes using ELISA. Also measurement of cellular immune responses through evaluation of spleen cells proliferation response using MTT and cytotoxicity by LDH were performed. Finally, the cytokine pattern of IFN-γ and IL-4 were also determined with ELISA. Results: The results indicate that candidate vaccine stimulated mouse splenic lymphocyte proliferation response and also induced strong cytotoxicity responses. Analysis of humoral immune response has shown that the candidate vaccine has induced specific antibody production mainly of the IgG2a sub-class. Also cytokine pattern evaluation has shown that IFN-γ secretion was dominant. Conclusion: The use of immunogen and conserved epitopes from P24 and Nef induced strong humoral and cellular immune responses and this construct could be candidate for further studies in animal models.

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Immunotoxins are an attractive way to treat cancer; in this method, high-cytotoxic protein toxins target cancer cells specifically. An immunotoxin consists of a targeting component (an antibody, cytokine, or other protein that binds to the cell), that is chemically conjugated or fused in DNA level to a cytotoxic cargo (a bacterium, plant or cytotoxic human protein). Immunotoxin, with the help of specific receptors, recognizes the target cell and enters the cell by endocytosis. After entering the cytocell, it kills the target cancer cell with the help of a toxic component. Although various immunotoxins with different structures have been studied and tested in recent decades, only three immunotoxins Denileukin Diftitox, Tagraxofusp and Moxestumomab Pasudotox - have been clinically approved for the treatment of leukemia. In this article, we review important research and two challenges in production and development of immunotoxins that have limited their clinical success. Further, we highlight methods to overcome these obstacles. These challenges include target and non-target cell toxicity and immunization.

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Objective: The objective of this study is to develop and assess targeted PAMAM-PEG nanocarrier with anti-TAG72 nanobody for t-Bid gene coding construct delivery into the human colonic adenocarcinoma cells. Materials and Methods: Nanobody (Nb) coding sequence was subcloned into pSJ expression vector for large-scale production and then Nb was purified by Ni++ affinity chromatography. SDS-PAGE and western blot analysis were performed to verify purifiction. PAMAM was reacted with PEG at the ratio 1:2 (mol/mol) and anti-TAG72 Nb at the ratio 1:1 (mol/mol). Surface charge and size of resulting nanoparticles were evaluated by Malvern zeta sizer and Nanosight. Efficiency of constructed gene carrier for t-Bid, a killer gene, delivery into colonic adenocarcinoma cells in in vitro was assessed using real time PCR and cell counting assays. Results: Production of nanoparticles with the average size of 162±92 nm and +4.57±0.52 zeta potential was confirmed by nanosight and Malvern zeta sizer in order. Gel retardation assay result verified efficiency of carrier for pDNA comlexation. Real time PCR results confirmed the target gene overexpression in the cancerous cell lines. Conclusion: The results of this research confirms the efficiency of PAMAM dendrimers for gene transferring, positive effect of PEGylation and targeting of nanoparticles by anti-TAG72 nanobody.

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Objective: Generation 5 poly (amidoamine) dendrimers are promising multipotent gene delivery vectors that provide favorable DNA condensation properties; however, their high toxicity limits their applications. Toxicity of PAMAM dendrimers depends on their type, generation and applied dosage in a way that lower generations (lower than G5 dendrimers) and anionic dendrimers have lower toxicity than higher generations and cationic dendrimers. The aim of this study is to evaluate the effect of PEGylation on toxicity of G5 PAMAM dendrimers. Methods: In this study, to improve their characteristics as gene delivery carriers, G5 PAMAM dendrimers were conjugated to polyethylene glycol molecules (PEG, molecular weight 3500) at three different molar ratios of 10, 20 and 30. Also the number of conjugated PEG chains was quantified using TNBSA and Ellman assays. The effect of different degrees of PEGylation on cytotoxicity and transfection efficiency of modified PAMAM dendrimers toward BT-474 and MCF-10A cell lines were assessed. Results: Compared to unconjugated, PEG conjugated PAMAM dendrimers had lower in vitro cytotoxicity, particularly at higher PEG to PAMAM molar ratios. Among all prepared PEG-PAMAM dendrimers, G5 PAMAM dendrimers that conjugated to PEG at a molar ratio of 10/1 had the highest in vitro transfection rate in both cell lines. Conclusion: Our results showed that these PEG-conjugated PAMAM dendrimers possess a great potential for in vitro gene delivery.

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Objective: Prostate cancer is the second cause of cancer-associated death in men. In recent years, targeted therapy for cancer has attracted the attention of researchers. Targeted therapy leads to a decrease in drug adverse effects. Studies indicate that targeting peptides for cancer cells represent valuable tools for diagnostics and therapeutics. Recently, phage display peptide libraries have been used to identify target peptides to a variety of cancer cells. In the current study, we aim to isolate peptides that target PC3 cells (human prostate adenocarcinoma cells). Methods: Four rounds of subtractive panning on control cells that included 5637 (bladder), Huh-7 (liver), SW480 (colon), AGS (stomach) and human fibroblast normal in addition to four rounds of positive panning on PC3 (target cell) were performed. Polyclonal phage ELISA was used to evaluate the process of enrichment during biopanning. Subsequently, phage clones were randomly selected from titer plates, amplified by plaque-PCR, and their genomic DNA was sequenced. We conducted bioinformatic analysis for further characterization of the isolated peptides. Results: Several rounds of panning resulted in the enrichment of a number of peptides. The results of polyclonal phage ELISA indicated that the biopanning process was successful. In silico analysis showed the presence of several consensus amino acid motifs in the peptides. Conclusion: The peptides identified through biopanning can be considered as potential specific binders to PC3 cells. Peptides with specificity binding to target cells can be used for targeted gene and drug delivery to malignant tumor cells. Further analyses of these peptides are required to show their capacity for targeted delivery of various genes and drugs into prostate cancer cells.

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Coronavirus disease 2019 (COVID-19) is a virally-induced pneumonia caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This is the third disease-causing coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and the middle east respiratory syndrome coronavirus (MERS-CoV) which has been known in the human population in the 21^st century. To this date (20^th of Mehr, 1399), more than thirty-four million people have been infected by this virus and more than a million have lost their lives because of it which further signifies the importance of COVID-19 prevention and treatment. In this review article, we first take a look at the history of the famous coronaviruses and then introduce the genetic and pathophysiology of SARS-CoV-2 in a detailed manner. After discussing the related clinical manifestations, we shed a light on the treatments that have been assessed to this date. In the end, we will briefly discuss the vaccines that are currency being developed and highlight their success rate, so far. It is delightful to assert that out own research team is currently developing an oral and/or respiratory vaccine against SARS-CoV-2 which is composed of spike-surface decorated chitosan nanoparticles.