Recombinant Core Protein Research Articles

Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia

BackgroundAlthough E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection.MethodsThe core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab−) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab+) samples that included viremic (chronic/relapsers) and recovered patients’ sera. The color intensity (OD450) and S/Co values were estimated.ResultsThe integration of 0.1–0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab−sera. The ideal S/Co threshold predicting active viremia (> 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection.ConclusionThe applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings.

Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for diagnosis of HCV infection in Egypt

BackgroundEgypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4. AimThis study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection. MethodsUsing synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid dilution method for further purification using weak cation exchange liquid chromatography. The immunogenicity of the purified protein was tested by ELISA using 129 serum samples. ResultsThe recombinant core protein was successfully expressed and purified. The results also showed that the in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the commercial ELISA kit. ConclusionThe sensitivity, specificity, and reproducibility of the developed assay were high and promising to be used as a screening assay for detecting HCV infection.

A simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies

In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.

Obtaining the VLPs from Recombinant Core Protein of Hepatitis C Virus (HCV)

Obtaining the VLPs from Recombinant Core Protein of Hepatitis C Virus (HCV)

IgG1 and IgG4 antibodies against Core and NS3 antigens of hepatitis C virus.

IgG subclasses involved in the immune response to hepatitis C virus (HCV) antigens have been rarely studied. We investigated the immune response mediated by IgG1 and IgG4 antibodies against the recombinant core and NS3 antigens in patients with chronic hepatitis C. Sixty patients infected with HCV genotype 1 without antiviral treatment and 60 healthy subjects participated in the study. Serum levels of alanine aminotransferase, HCV viremia, and the presence of cryoglobulinemia and liver fibrosis were determined. We investigated the serum IgG1 and IgG4 antibodies against recombinant HCV core and NS3 non-structural protein antigens using amplified indirect ELISA. Anti-core and anti-NS3 IgG1 antibodies were detected in 33/60 (55%) and 46/60 (77%) patients, respectively, whereas only two healthy control samples reacted with an antigen (NS3). Anti-core IgG4 antibodies were not detected in either group, while 30/60 (50%) patients had anti-NS3 IgG4 antibodies. Even though there were higher levels of anti-NS3 IgG4 antibodies in patients with low viremia (< 8 × 105 IU/mL), IgG1 and IgG4 antibody levels did not correlate with ALT levels, the presence of cryoglobulinemia, or degree of hepatic fibrosis. High production of anti-core and anti-NS3 IgG1 antibodies was observed in chronic hepatitis C patients. In contrast, IgG4 antibodies seemed to only be produced against the NS3 non-structural antigen and appeared to be involved in viremia control. IgG1 antibodies against structural and non-structural antigens can be detected in chronic hepatitis C, while IgG4 antibodies seem to be selectively stimulated by non-structural HCV proteins, such as the NS3 antigen.

Xylosylation of proteins by expression of human xylosyltransferase 2 in plants

Through the years, the post-translational modification of plant-made recombinant proteins has been a considerable problem. Protein glycosylation is arguably the most important post-translational modification; thus, for the humanization of protein glycosylation in plants, the introduction, repression, and knockout of many glycosylation-related genes has been carried out. In addition, plants lack mammalian-type protein O-glycosylation pathways; thus, for the synthesis of mammalian O-glycans in plants, the construction of these pathways is necessary. In this study, we successfully xylosylated the recombinant human proteoglycan core protein, serglycin, by transient expression of human xylosyltransferase 2 in Nicotiana benthamiana plants. When human serglycin was co-expressed with human xylosyltransferase 2 in plants, multiple serine residues of eight xylosylation candidates were xylosylated. From the results of carbohydrate assays for total soluble proteins, some endogenous plant proteins also appeared to be xylosylated, likely through the actions of xylosyltransferase 2. The xylosylation of core proteins is the initial step of the glycosaminoglycan part of the synthesis of proteoglycans. In the future, these novel findings may lead to whole mammalian proteoglycan synthesis in plants.

Structural characterization of the HCoV-229E fusion core

HCoV-229E spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This protein is composed of an N-terminal receptor-binding domain (S1) and a C-terminal trans-membrane fusion domain (S2). S2 contains a highly conserved heptad repeat 1 and 2 (HR1 and HR2). In this study, the HRs sequences were designed and connected with a flexible linker. The recombinant fusion core protein was crystallized and its structure was solved at a resolution of 2.45 Å. Then we characterized the binding of HR1s and HR2s via both sequence alignment and structural analysis. The overall structures, especially the residues in some positions of HR2 are highly conserved. Fourteen hydrophobic and three polar residues from each HR1 peptide are packed in layers at the coiled-coil interface. These core amino acids can be grouped into seven heptad repeats. Analysis of hydrophobic and hydrophilic interactions between HR2 helix and HR1 helices, shows that the HR1 and HR2 polypeptides are highly complementary in both shape and chemical properties. Furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting membrane fusion, a crucial step of HCoV-229E infection.

Hepatitis C virus core protein reduces CD8+ T-cell proliferation, perforin production and degranulation but increases STAT5 activation.

Clearance of hepatitis C virus (HCV) is dependent on an effective virus-specific CD8+ T-cell response, which is dysfunctional in chronic HCV infection. Dysfunction in bulk or non-HCV-specific CD8+ T-cells in HCV infection has also been observed. This may contribute to observed reductions in immunity to other diseases (e.g. cancer, viral co-infections) in HCV-infected individuals. Evidence suggests that the HCV core protein (found in blood as free protein) may contribute to this impairment. To determine if HCV core contributes to the impairment of effector functions and survival potential of CD8+ T-cells, isolated human CD8+ T-cells from healthy donors were pre-incubated with recombinant HCV core protein for 72 hr and then stimulated in vitro to evaluate proliferation, survival potential and effector functions. Pre-incubation of stimulated CD8+ T-cells with HCV core significantly reduced their proliferation. Perforin production and degranulation were also decreased, but interferon-γ production was unchanged. Additionally, when CD8+ T-cells were treated with serum from HCV+ individuals, they produced less perforin than cells treated with healthy serum. Up-regulation of anti-apoptotic Bcl-2 was slightly lower in cells treated with HCV core, but signal transducer and activator of transcription 5 (STAT5) activation was increased, suggesting dysregulation downstream of STAT activation. Our study reveals that HCV core reduces the activity and target lysis-associated functions of CD8+ T-cells. This may contribute to the generalized impairment of CD8+ T-cells observed in HCV infection. These findings provide insight for the design of novel counteractive immune-mediated strategies including the design of effective therapeutic vaccines for use in HCV+ individuals.

Defects in chondrocyte maturation and secondary ossification in mouse knee joint epiphyses due to Snorc deficiency.

The role of Snorc, a novel cartilage specific transmembrane proteoglycan, was studied during skeletal development using two Snorc knockout mouse models. Hypothesizing that Snorc, like the other transmembrane proteoglycans, may be a coreceptor, we also studied its interaction with growth factors. Skeletal development was studied in wild type (WT) and Snorc knockout mice during postnatal development by whole mount staining, X-ray imaging, histomorphometry, immunohistochemistry and qRT-PCR. Snorc promoter activity was studied by applying the LacZ reporter expressed by the targeting construct. Slot blot binding and cell proliferation assays were used to study the interaction of Snorc with several growth factors. Snorc expression was localized in the knee epiphyses especially to the prehypertrophic chondrocytes delineating the cartilage canals and secondary ossification center (SOC). Snorc was demonstrated to have a glycosaminoglycan independent affinity to FGF2 and it inhibited FGF2 dependent cell growth of C3H101/2cells. In Snorc deficient mice, SOCs in knee epiphyses were smaller, and growth plate (GP) maturation was disturbed, but total bone length was not affected. Central proliferative and hypertrophic zones were enlarged with higher extracellular matrix (ECM) volume and rounded chondrocyte morphology at postnatal days P10 and P22. Increased levels of Ihh and Col10a1, and reduced Mmp13 mRNA expression were observed at P10. These findings suggest a role of Snorc in regulation of chondrocyte maturation and postnatal endochondral ossification. The interaction identified between recombinant Snorc core protein and FGF2 suggest functions related to FGF signaling.

INDUCTION OF T-CELL IMMUNE RESPONSE IN CHRONIC HCV-INFECTED PATIENTS WITH UNDERLYING DENDRITIC CELL IMMUNOTHERAPY

A key role of T cells in viral elimination and absence of strong T cell responses in patients with chronic hepatitis C virus (HCV) infection presumes that activation of antigen-specific T cells may be a promising approach to enhance treatment efficacy. Given the central role of dendritic cells (DCs) in the induction of T cell response, the aim of our study was to evaluate effects of DC immunotherapy upon immunological parameters in chronic HCV infection. Ten patients with chronic hepatitis C (genotype 1) were vaccinated with monocytederived DCs, generated in presence of IFNα (IFN-DCs) and pulsed with recombinant HCV Core (1–120) and NS3 (1192–1457) proteins. The vaccination protocol included as initiating procedure (one injection per week, ns = 4) and maintaining treatment (one monthly injection, ns = 6), with subsequent follow up for 6 months. The immunotherapy was not associated with serious adverse events, significant post-vaccination reactions, or increased hepatitis C activity, according to biochemical tests. Ex vivo studies have shown that immunotherapy elicited strong and stable immune response to Core and moderate response to NS3 protein, which manifested as a significant increase of MNC proliferation and IFNγ production in response to Core and enhancement of IFNγ production (without higher proliferation rates), in response to NS3. DC immunotherapy also led to increase of ConA-induced MNC proliferation up to normal levels indicating a recovery of mitogenic T cell reactivity. Meanwhile, T cell activation did not elicit antigen-specific Th2 response and expansion of CD4 + CD25 + CD127 regulatory T cells. Despite induced immune response, the immunotherapy with DCs was not accompanied by decreased viremia levels. Nevertheless, a transitory decrease of viral load in four patients and stable decrease of viremia in two patients as well as an inverse correlation between NS3-specific proliferation and viremia (Rss = 0.62; p < 0.05) by the end of 6-month follow-up indicated that the antigenspecific T cells may have a potential to control viral replication.

Clinical Evaluation of Terap C Vaccine in Combined Treatment with Interferon and Ribavirin in Patients with Hepatitis C

BackgroundAn estimated 170 million individuals worldwide are infected with the hepatitis C virus (HCV). Although treatment options using a combination of pegylated interferon and ribavirin (P-IFN/RBV) are available, sustained clearance of the virus is only achieved in approximately 40% of individuals infected with HCV genotype 1. Recent advances in the treatment of HCV using directly acting antiviral agents have been achieved; however, treatment can be very expensive and is associated with substantial side effects. The development of a new treatment modality is needed. One possible modality could be specific immunotherapy. Terap C is a therapeutic vaccine candidate composed of pIDKE2, a plasmid expressing HCV structural antigens, with a recombinant HCV core protein, Co.120. ObjectiveTo assess the safety and efficacy of concomitant therapy with the candidate vaccine, Terap C, IFN α-2b and ribavirin in untreated individuals with HCV genotype 1 infection. MethodsThis was a Phase II randomized, placebo-controlled, double-blind clinical trial evaluating the safety and efficacy of Terap C concomitant with IFN α-2b/RBV in 92 treatment-naïve patients with HCV genotype 1 infection. The study was conducted at the Gastroenterology Institute in Havana, Cuba. Patients were randomly assigned to 1 of 5 groups. The control group (Group 1) received IFN α-2b/RBV and placebo for 48 weeks. Groups 2 and 3 were administered Terap C 6 and 9 times, respectively, in addition to standard IFN α-2b/RBV treatment. In groups 4 and 5, Terap C was introduced 12 weeks after the initiation of IFN α-2b/RBV and administered 6 and 9 times, respectively, concomitant with IFN α-2b/RBV. ResultsAll patients showed some adverse events. Out of 3615 adverse events, only 18.8% were considered to be probably associated with administration of Terap C. Most events (47.4%) were considered to be improbably associated with of administration Terap C. Only 33.8% were considered possibly temporarily associated with Terap C, and can be explained by the use of conventional IFN α-2b + RBV or by HCV itself. The most common adverse events (≥65%) observed were pain at the injection site, headache, asthenia, psychiatric disturbances, fever, and gastrointestinal symptoms. Regarding sustained virological response, a 20% superiority was observed in the patients who received concomitant Terap C treatments from the beginning of the study compared with those who started after Week 12. ConclusionsVaccination with Terap C in patients with chronic HCV infection was safe and well tolerated. Clinical trial protocol code: IG/VHI/HC/0701; Public Register Code: RPCEC00000074.

The cytoprotective effect of biglycan core protein involves Toll-like receptor 4 signaling in cardiomyocytes

AimsExogenously administered biglycan (core protein with high-molecular weight glycosaminoglycan chains) has been shown to protect neonatal cardiomyocytes against simulated ischemia/reperfusion injury (SI/R), however, the mechanism of action is not clear. In this study we aimed to investigate, which structural component of biglycan is responsible for its cardiocytoprotective effect and to further explore the molecular mechanisms involved in the cytoprotection. Methods and resultsA pilot study was conducted to demonstrate that both native (glycanated) and deglycanated biglycan can attenuate cell death induced by SI/R in a dose-dependent manner in primary neonatal cardiomyocytes isolated from Wistar rats. In separate experiments, we have shown that similarly to glycanated biglycan, recombinant human biglycan core protein (rhBGNc) protects cardiomyocytes against SI/R injury. In contrast, the glycosaminoglycan component dermatan sulfate had no significant effect on cell viability, while chondroitin sulfate further enhanced cell death induced by SI/R. Treatment of cardiomyocytes with rhBGNc reverses the effect of SI/R upon markers of necrosis, apoptosis, mitochondrial membrane potential, and autophagy. We have also shown that pharmacological blockade of Toll-like receptor 4 (TLR4) signaling or its downstream mediators (IRAK1/4, ERK, JNK and p38 MAP kinases) abolished the cytoprotective effect of rhBGNc against SI/R injury. Pretreatment of cardiomyocytes with rhBGNc for 20h resulted in increased Akt phosphorylation and NO production without having significant effect on phosphorylation of ERK1/2, STAT3, and on the production of superoxide. Treatment over 10min and 1h with rhBGNc increased ERK1 phosphorylation, while the SI/R-induced increase in superoxide production was attenuated by rhBGNc. Blockade of NO synthesis also prevented the cardiocytoprotective effect of rhBGNc. ConclusionsThe core protein of exogenous biglycan protects myocardial cells from SI/R injury via TLR4-mediated mechanisms involving activation of ERK, JNK and p38 MAP kinases and increased NO production. The cytoprotective effect of rhBGNc is due to modulation of SI/R-induced changes in necrosis, apoptosis and autophagy.

Electrophysiological Characterization of two Novel Ion Channels of Mitochondria

Mitochondrial ion channels are of great importance to ensure the proper function of this bioenergetic organelle and to regulate cell fate. However, in many cases our knowledge concerning their molecular identity and regulation is still limited. Here we describe a novel regulatory mechanism of the voltage-dependent anion channel VDAC3 and a new channel-forming protein. Voltage-dependent anion channels (VDAC), also known as eukaryotic porins, are located in the outer mitochondrial membrane and allow the flux of ions and small metabolites. While the pore-forming ability of recombinant VDAC1 and VDAC2 has been extensively studied during the last decades, a clear-cut ion conducting channel activity has been assigned to the VDAC3 isoform only recently (Checchetto et al, Cell Phys Biochem, 2014). This protein forms an ion channel with small conductance under standard conditions, but our study identifies the amino acid residues whose oxidation state impacts on channel activity and conductance. Furthermore, we characterize from electrophysiological point of view a novel protein complex composed by pore-forming core and regulatory subunits located in the inner mitochondrial membrane. Channel activity can be observed with the reconstituted recombinant core protein, but for its regulation the co-expression of both the core and the regulatory subunits are necessary. Overall, our data suggest that VDAC3 and the novel protein complex are new players in the control of mitochondrial ion homeostasis and might contribute to the plasticity of mitochondrial function in intact cells.

Inhibitory effect of miR-125b on hepatitis C virus core protein-induced TLR2/MyD88 signaling in THP-1 cells.

To investigate the role of miR-125b in regulating monocyte immune responses induced by hepatitis C virus (HCV) core protein. Monocytic THP-1 cells were treated with various concentrations of recombinant HCV core protein, and cytokines and miR-125b expression in these cells were analyzed. The requirement of Toll-like receptor 2 (TLR2) or MyD88 gene for HCV core protein-induced immune responses was determined by the transfection of THP-1 cells with gene knockdown vectors expressing either TLR2 siRNA or MyD88 siRNA. The effect of miR-125b overexpression on TLR2/MyD88 signaling was examined by transfecting THP-1 cells with miR-125b mimic RNA oligos. In response to HCV core protein stimulation, cytokine production was up-regulated and miR-125b expression was down-regulated in THP-1 cells. The modulatory effect of HCV core protein on cellular events was dose-dependent and required functional TLR2 or MyD88 gene. Forced miR-125b expression abolished the HCV core protein-induced enhancement of tumor necrosis factor-α, interleukin (IL)-6, and IL-10 expression by 66%, 54%, and 66%, respectively (P < 0.001), by inhibiting MyD88-mediated signaling, including phosphorylation of NF-κBp65, ERK, and P38. The inverse correlation between miR-125b and cytokine expression after HCV core challenge suggests that miR-125b may negatively regulate HCV-induced immune responses by targeting TLR2/MyD88 signaling in monocytes.

Different expression systems for production of HCV structural proteins

Hepatitis C virus (HCV) is an important agent causing chronic liver infection, which often leads to liver cirrhosis and lethal hepatocellular carcinoma (HCC). At present, there is no effective HCV vaccine for prevention of hepatic disease and the standard treatment is neither economical nor fully effective in all the patients. However, vaccination based on structural and nonstructural proteins of HCV has attracted a special interest. Different heterologous systems have been used to generate the recombinant HCV core, E1, and E2 proteins including Escherichia coli , yeast, insects and mammalian cells. Further studies showed that the amounts of HCV recombinant proteins in E. coli are more suitable and un-expensive compared to other systems. It should be considered that this system is not efficient for generation of the glycosylated proteins. Thus, the structure of proteins is an important agent of selection for expression systems. The selection of expression systems will be critical for the use of recombinant proteins as an immunogen. In this mini-review, we briefly describe different expression systems for generation of the HCV recombinant structural proteins applied in vaccine design.

Heterologous Expression of Hepatitis C Virus Core Protein in Oil Seeds of Brassica napus L.

Background:Hepatitis c virus (HCV), prevalent among 3% of the world population, is a major worldwide public health concern and an effective vaccination could help to overcome this problem. Plant seeds as low-cost vaccine expression platforms are highly desirable to produce antigens.Objectives:The present study was aimed at investigating the possible expression of recombinant HCV core protein, as a leading HCV vaccine candidate, in canola (Brassica napus) plant seeds in order to be used as an effective immunogen for vaccine researches.Materials and Methods:A codon-optimized gene harboring the Kozak sequence, 6 × His-tag, HCVcp (1 - 122 residues) and KDEL (Lys-Asp-Glu-Leu) peptide in tandem was designed and expressed under the control of the seed specific promoter, fatty acid elongase 1 (FAE1), to accumulate the recombinant protein in canola (B. napus L.) seeds. Transgenic lines were screened and the presence of the transgene was confirmed in the T0 plants by polymerase chain reaction (PCR). The quantity and quality of the HCV core protein (HCVcp) in transgenic seeds were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot, respectively.Results:Western blot analysis using anti-His antibody confirmed the presence of a 15 kDa protein in the seeds of T1 transgenic lines. The amount of antigenic protein accumulated in the seeds of these transgenic lines was up to 0.05% of the total soluble protein (TSP).Conclusions:The canola oilseeds could provide a useful expression system to produce HCV core protein as a vaccine candidate.

Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

Expression and Purification of HCV Core and Core-E1E2 Proteins in Different Bacterial Strains.

Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses. In current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems. The expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer. The data showed a clear band of ~573 bp for HCV core and ~2238 bp for core-E1E2 genes in agarose gel. Moreover, a ~21 kDa band of core protein and a ~83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method. This study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system.

Different protein expression systems can influence the direction of the immune responses against HCV core protein in animal model

Introduction: Hepatitis C virus (HCV) infection is a major public health problem which influences about 170 million people worldwide. Different types of vaccines have been designed using HCV structural proteins to control the viral infection. The core nucleocapsid protein is one of the most conserved proteins and a desirable target for HCV vaccines, especially the protein-based vaccines. In current study, we generated the core protein recombinantly in prokaryotic (Escherichia coli) and eukaryotic (Leishmania tarentolae) expression systems and compared the humoral immune responses stimulated by each protein in a BALB/c mice model. Methods: The expression of HCV core protein was performed using the prokaryotic pET-28a/ BL21 expression system and also the eukaryotic LEXSY expression system. The recombinant core proteins expressed in E. coli and Leishmania were purified using reverse staining method and affinity chromatography under native conditions, respectively. The purified core proteins were detected by SDS-PAGE and Western blotting using anti-His antibody and were assessed by NanoDrop spectrophotometer. Finally, the abilities of both recombinant core proteins to induce the effective humoral immune responses were evaluated using indirect ELISA. Results: Our data indicated a clear band of ~ 21 kDa for the purified HCV core proteins in both expression systems, confirmed by SDS-PAGE and Western blotting. The mice immunization with both recombinant core proteins was able to produce high levels of antibody isotypes (IgG1 and IgG2a) in comparison with the controls (p < 0.05). In addition, the level of IgG2a response was significantly higher in the group immunized with the core protein purified from leishmania compared to the protein generated from E. coli (p < 0.05). Conclusion: The recombinant core protein generated by the leishmania expression system could induce a Th1-biased immune response with respect to the increase of IgG2a to IgG1 ratio.

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C.