Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients

Objective(s): Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3) of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3) using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3). The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: The insertion of the DNA fragment of the NS3 region into the expression vector was further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy. © 2015, Mashhad University of Medical Sciences. All rights reserved

Establishment of a new immunological method for direct detection of Mycobacterium in solution

Background/PurposeTuberculosis (TB) is a crucial health problem. Prevention of the disease requires rapid diagnosis. Rapid liquid culture systems, nucleic acid amplification tests, and high-performance liquid chromatography (HPLC) are among the rapid tests used for detecting Mycobacterium species. However, these tests are expensive and require extensive equipment and expertise, which is hardly affordable in resource-poor countries. Although direct microscopy is performed routinely as an initial step for detection of the bacteria, it is not sufficiently sensitive. As a result, we thought of establishing a low-cost immunological test that can potentially replace direct microscopy with higher sensitivity and specificity.MethodsThe assay is based on pre-incubation of biotinylated rabbit antibody against Antigen 60 (A60) with a solution containing Bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis (MTB) followed by incubation with a streptavidin–alkaline phosphatase (STA–ALP) conjugate. The test is devised in enzyme-linked immunosorbent assay (ELISA) and non-ELISA formats, therefore it does not require extensive facilities and expertise.ResultsThe ELISA format showed a 100-fold improvement in the lower detection limit of BCG compared with direct microscopy. With the non-ELISA formats, there was a 2- and 16-fold improvement for the cartridge assay and the microfuge tube assay, respectively.ConclusionIn conclusion, we successfully detected BCG and MTB in solution using the new immunological method. Our results are very promising and the new immunological method could potentially replace direct microscopy with higher sensitivity and specificity

Analysis of immumoreactivity of heterologously expressed non-structural protein 4B (NS4B) from Hepatitis C Virus (HCV) genotype 1a

Background: Detection of hepatitis C virus specific antibodies is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and has been widely used in commercial Enzyme Immunoassays (EIA). Additionally, NS4B, a key protein in the virus replication, can be an alternative target for antiviral therapy. Objectives: Development of a new method for high-level expression and purification of NS4B coding region was the aim of the report. Materials and Methods: Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. coli BL21. Induction was performed by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. Immunoreactivity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzymelinked immunosorbent assay (ELISA). Results: The recombinant NS4B protein was expressed and its immunoreactivity was confirmed by ELISA and western blotting. Conclusions: The directional TOPO cloning provides an efficient and easy platform for heterologous expression of immunoreactiveHCV NS4B. © 2015 Kowsar Medical Publishing Company. All rights reserved

Study of NGEP expression in androgen sensitive prostate cancer cells: A potential target for immunotherapy

Background: Prostate cancer is one of the leading causes of cancer deaths among men. New gene expressed in prostate (NGEP), is a prostate-specific gene expressed only in normal prostate and prostate cancer tissue. Because of its selective expression in prostate cancer cell surface, NGEP is a potential immunotherapeutic target. To target the NGEP in prostate cancer, it is essential to investigate its expression in prostate cancer cells. Methods: In the present study, we investigated NGEP expression in LNCaP and DU145 cells by real time and RT-PCR, flow cytometric and immunocytochemical analyses. Results: Real time and RT-PCR analyses of NGEP expression showed that NGEP was expressed in the LNCaP cells but not in DU145 cells. The detection of NGEP protein by flow cytometric and immunocytochemistry analyses indicated that NGEP protein was weakly expressed only in LNCaP cell membrane. Conclusion: Our results demonstrate that LNCaP cell line is more suitable than DU145 for NGEP expression studies; however, its low-level expression is a limiting issue. NGEP expression may be increased by androgen supplementation of LNCaP cell culture medium

Leucine-rich amelogenin peptide (LRAP) as a surface primer for biomimetic remineralization of superficial enamel defects: An in vitro study

This study was carried out to obtain more information about the assembly of hydroxyapatite bundles formed in the presence of Leucine-Rich Amelogenin Peptide (LRAP) and to evaluate its effect on the remineralization of enamel defects through a biomimetic approach. One or 2 mg/mL LRAP solutions containing 2.5 mM of Ca+2 and 1.5 mM phosphate were prepared (pH = 7.2) and stored at 37 °C for 24 h. The products of the reaction were studied using atomic force microscopy (AFM), transmission electron microscopy (TEM), and selected area electron diffraction (SAED). Vickers surface microhardness recovery (SMR) of acid-etched bovine enamel, with or without LRAP surface treatment, were calculated to evaluate the influence of peptide on the lesion remineralization. Distilled water and 1 or 2 mg/mL LRAP solution (pH = 7.2) were applied on the lesions and the specimens were incubated in mineralization solution (2.5mM Ca+2, 1.5mM PO4 -3, pH = 7.2) for 24 h. One-way ANOVA and Tukey's multi-comparison tests were used for statistical analysis. The pattern of enamel surface repair was studied using FE-SEM. AFM showed the formation of highly organized hierarchical structures, composed of hydroxyapatite (HA) crystals, similar to the dental enamel microstructure. ANOVA procedure showed significant effect of peptide treatment on the calculated SMR (p < 0.001). Tukey's test revealed that peptide treated groups had significantly higher values of SMR. In conclusion, LRAP is able to regulate the formation of HA and enhances the remineralization of acid-etched enamel as a surface treatment agent. © 2015 Wiley Periodicals, Inc

In-vitro prostate cancer biomarker detection by directed conjugation of anti-PSCA antibody to super paramagnetic iron oxide nanoparticless

Background: The main property of a successful conjugation of antibodies to nanoparticles is keeping the potency of antibody for binding the antigen, and an oriented conjugation can do that. Under such ground, this study was carried out to explore the efficiency of two conjugation methods in binding iron nanoparticles to an antibody produced against PSCA (prostate stem cell antigen) using in vitro labeling of PC3 cells. Methods: In this experimental study, we conjugated dextran-superparamagnetic iron oxide nanoparticles (dexSPIONs) to anti-PSCA antibody by two different methods, including targeting carbohydrate moieties in FC domain and the free amine group of amino acid side chains. Ultimately, Iron staining was done by anti-PSCA antibody-dexSPIONs in PC3 cells to detect antibody binding to the cells. Results: A strong blue dye was induced by iron staining in conjugated dexSPIONs on the membrane of PC3 cells by the former method than the second one. Moreover, cells treated with 20 nm diameters of dexSPIONs showed higher resolution of blue color than those treated with 100 nm nanoparticles. Conclusion: This oriented conjugation method promoted the efficiency of targeting tumor antigens, and the presence of iron particles might enhance MRI image intensity in vivo by targeting PSCA-overexpressing cells in future studies. © Iran University of Medical Sciences

Designing a chimeric subunit vaccine for influenza virus, based on HA2, M2e and CTxB: a bioinformatics study

Background: Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either. Results: Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine. Conclusions: It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant. © 2020, The Author(s)

Preparation of immunotoxin herceptin-botulinum and killing effects on two breast cancer cell lines

Background: Worldwide, breast cancer is the most common cancer diagnosed among women and a leading cause of cancer deaths. The age of onset in Iran has become reduced by a decade for unknown reasons. Herceptin, a humanized monoclonal antibody, is a target therapy for breast cancer cells with over expression of HER2-neu receptors, but it is an expensive drug with only 20 beneficial rate of survival. This study introduces a novel approach to enhance the efficacy of this drug through immunoconjugation of the antibody to botulinum toxin. Decreasing the cost and adverse effects of the antibody were secondary goals of this study. Materials and Methods: Botulinum toxin was conjugated with Herceptin using heterobifunctional cross linkers, succinimidyl acetylthiopropionate (SATP) and sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the supplier's guidelines and tested on two breast cancer cell lines: SK-BR-3 and BT-474. Toxin and Herceptin were also used separately as controls. The cytotoxicity assay was also performed using the new bioconjugate on cultured cells with Alamar blue and a fluorescence plate reader. Results: Herceptin-Toxin bioconjugation significantly improved Herceptin efficacy on both breast cancer cell lines when compared to the control group. Conclusions: Toxin-Herceptin bioconjugation can be a potential candidate with increased efficiency for treating breast cancer patients with over expression of the HER2 receptor

The level of aflatoxin M1 in raw and pasteurized milk produced in alborz province, Iran

Background: Aflatoxins are agroupof very toxiccompoundsproducedby a fungal speciesandare foundin food products. Aflatoxin M1 (AFM1) is a type found in dairy products and resistant to pasteurization. AFM1 can adversely affect hepatocytes in human and leading to various liver diseases. Thus, it is essential to examine raw milk for the presence of AFM1. Objectives: The current study aimed to evaluate the levels of AFM1 in raw and pasteurized milk produced in Alborz province, Iran. Materials and Methods: Ninety seven samples of rawmilk and 20 samples of pasteurized milk were collected from milk collecting centers and different supermarkets, respectively. Samples were analyzed to determine AFM1 level by immunoaffinity column chromatography, and high-performance liquid chromatography (HPLC) using a C18 column with fluorescence detector. Mobile phase was water-acetonitrile-methanol (6:2:2 V/V/V) at a flow rate of 1.0 mL/minute. Results: AFM1 was detected in all samples with various concentration levels ranging from 0.0024 to 0.231 μg/kg, the mean concentration = 0.027±0.018 μg/kg. Conclusions: According to the results of the current study, 9.27 of the rawmilk and 5 of the pasteurized milk samples had higher levels of AFM1 than the maximum recommended limit (0.05 μg/kg) by food and agriculture organization (FAO). © 2016, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences

Improving proteome coverage for HS578T breast cancer cell-line due to efficient interfering removal

Background: In spite of the wide use, low proteome coverage and fuzzy patterns are the most important deterrents for the clinical applications of gel-based proteomic studies. So herein, we tried to increase the 2-dimentional proteome coverage of Hs578T breast cancer cells via investigating the efficacy of the three common techniques, usually used for interfering removal. Methods: Hs578T cells were incubated in a lysis solution to obtain raw cell extracts. Cellular soups of each extraction were then pooled, homogenized, and aliquoted to be further treated by three different protein-specific purification methods including acetone, acetone-methanol, and trichloroacetic acid (TCA)-acetone, each in triplicates. All the purified protein pellets were then dissolved in a standard rehydration buffer solution, loaded into the 17-cm immobilized pH gradient (IPG) strips, and separated according to their isoelectric points. Proteins were then separated once more according to their molecular weights in an O'Farrell separation system. Finally, by the visualization of the protein spots on the 2-dimentional profiles, quality and quantity of these 2-dimentional proteome patterns were then analyzed using the ImageMaster software. Findings: The obtained proteome recovery yields and total protein counts for acetone, acetone-methanol, and trichloroacetic acid-acetone methods were 0.100 ± 0.001, 0.070 ± 0.002, and 0.120 ± 0.005 ng/cell, and 1299 ± 9, 1698 ± 14 and 1973 ± 17, respectively. The results represent data obtained from three independent experiments. Conclusion: Trichloroacetic acid-acetone purification not only represented the highest recovery yield, suitable for expensive assays, but also showed the most suitable proteome coverage. So, the method is recommended for the comparative proteomic studies. However, the acetone-methanol procedure is more recommended for serological proteome analysis (SERPA); since it represents stronger protein spots which are more fitted to the immunoblotting procedure. © 2018, Isfahan University of Medical Sciences(IUMS). All rights reserved