RESUMEN
Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263 T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43 kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50 °C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. Km and kcat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22 mM and 417.1 min-1, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.
Asunto(s)
Proteínas Bacterianas , Epítopos , Geobacillus/genética , Lipasa , Proteínas Recombinantes de Fusión , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Epítopos/biosíntesis , Epítopos/química , Epítopos/genética , Epítopos/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Geobacillus/enzimología , Lipasa/biosíntesis , Lipasa/química , Lipasa/genética , Lipasa/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa , Campylobacter coli , Campylobacter jejuni , Proteínas Portadoras , Epítopos , Expresión Génica , Proteínas Recombinantes de Fusión , Animales , Anticuerpos Antibacterianos/química , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Campylobacter coli/química , Campylobacter coli/genética , Campylobacter coli/inmunología , Campylobacter jejuni/química , Campylobacter jejuni/genética , Campylobacter jejuni/inmunología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Epítopos/biosíntesis , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Conejos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The swine pathogen porcine circovirus type 2 (PCV2) causes significant economic damage worldwide. The PCV2 capsid (CP) residues 169-STIDYFQPNNKR-180 have been identified as a decoy epitope that diverts the host immune response away from protective epitopes. However, the decoy epitope may include important linear or conformational protective epitopes against PCV2. In this study, we used the baculovirus system to express recombinant complete CP (1-233) and mutant CP (Δ169-180), in which the decoy epitope was deleted, and evaluated the immune response to these in mice. Immunization with mutant CP (Δ169-180) protein, which formed very low level of virus-like particles (VLPs), elicited significantly lower levels of PCV2 CP-specific IgG antibodies and a slightly lower neutralizing activity than immunization with the complete CP (1-233) protein. This finding suggests that the complete CP is important for efficient VLP assembly and induction of PCV2-specific IgG antibodies and neutralizing antibodies in mice. This study may provide useful information for next-generation vaccine design for PCV2 control.
Asunto(s)
Proteínas de la Cápside/inmunología , Circovirus/inmunología , Epítopos/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/análisis , Anticuerpos Antivirales/análisis , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Circovirus/genética , Epítopos/biosíntesis , Epítopos/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Síndrome Multisistémico de Emaciación Posdestete Porcino/inmunología , Síndrome Multisistémico de Emaciación Posdestete Porcino/prevención & control , Porcinos , Vacunación , Vacunas de Partículas Similares a Virus/genéticaRESUMEN
Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients' sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.
Asunto(s)
Antígenos Virales , Virus del Dengue/genética , Epítopos , Expresión Génica , Proteínas Recombinantes de Fusión , Proteínas del Envoltorio Viral , Virus Zika/genética , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Baculoviridae/genética , Baculoviridae/metabolismo , Línea Celular , Epítopos/biosíntesis , Epítopos/genética , Mariposas Nocturnas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genéticaRESUMEN
Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Leones/inmunología , Tigres/inmunología , Células Epiteliales Alveolares/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos/inmunología , Células CHO , Gatos , Bovinos , Cricetinae , Cricetulus , Mapeo Epitopo , Epítopos/biosíntesis , Caballos/inmunología , Humanos , Glicoproteínas de Membrana/inmunología , Ratones , Podocitos/inmunología , Conejos , Ratas , Ovinos/inmunología , Porcinos/inmunologíaRESUMEN
An antisheep podoplanin (sPDPN) monoclonal antibody (mAb), PMab-256, has recently been established. PMab-256 shows positive immunostaining for lymphatic endothelial cells, lung type I alveolar cells, and kidney podocytes. PDPN possesses three platelet-aggregation-stimulating (PLAG) domains, PLAG1, PLAG2, and PLAG3, and a PLAG-like domain (PLD). The binding epitope of many anti-PDPN mAbs is located in PLAG domains or PLD. The purpose of this study is to determine the binding epitope of PMab-256. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-256 epitope exists between amino acids 75 and 80 of sPDPN. Furthermore, analysis of sPDPN point mutations demonstrated that the critical epitope includes Thr80 of sPDPN, indicating that the PMab-256 epitope is in the PLD of sPDPN.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Glicoproteínas de Membrana/inmunología , Ovinos/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos/inmunología , Células CHO , Cricetinae , Cricetulus , Células Endoteliales/inmunología , Mapeo Epitopo , Epítopos/biosíntesis , Humanos , Podocitos/inmunologíaRESUMEN
BACKGROUND: The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera. OBJECTIVE: The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1). METHODS: For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures. RESULTS: Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study. CONCLUSION: This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.
Asunto(s)
Epítopos , Virus Linfotrópico T Tipo 1 Humano/genética , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión , Proteínas Virales , Epítopos/biosíntesis , Epítopos/química , Epítopos/genética , Humanos , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Virales/biosíntesis , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
OBJECTIVE: To analyse the immunogenicity of a fusion protein containing cell epitopes of Mycobacterium tuberculosis genes Rv2660c, Rv2460c, Rv3875 and Rv3804, and to evaluate the feasibility of using it as a novel target antigen for developing multi-stage TB vaccines. METHODS: Cell epitopes of Rv2660c, Rv2460c, Rv3875 and Rv3804c were fused in series to form a new antigen gene (named msv). Then msv was cloned into the prokaryotic expression vector pEASY-Blunt E1. The fusion protein msv was expressed by pEASY-Blunt E1 under the induction of isopropyl-ß-d-thiogalactoside (IPTG). Purified the protein by affinity chromatography and identified the protein by SDS-PAGE and Western blot. To evaluate the immunogenicity of the protein, the mice were immunized with the purified fusion protein, and the titer of the antibody in mice serum was evaluated by ELISA. Besides, splenocytes of immunized mice were separated and splenocytes proliferation was determined under the stimulation of the protein. RESULTS: The prokaryotic expression plasmid carrying msv gene was constructed successfully and msv protein could be expressed by the plasmid under the induction of IPTG. SDS-PAGE and Western blot results confirmed that a purified protein (relative molecular mass was 41.3×103) was obtained. ELISA result indicated that the titer of the antibody in msv immunized mice serum was about 1:81 920.The spleen lymphocyte proliferation assay showed that after immunization with msv protein, significant proliferation of antigen-sensitized lymphocytes was observed. CONCLUSIONS: The fusion protein msv was successfully expressed and purified, which can induce humoral and cellular immunity in mice. It may be used as an antigen component for the development of TB vaccine in the future.
Asunto(s)
Proteínas Bacterianas/inmunología , Epítopos/biosíntesis , Mycobacterium tuberculosis , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Antígenos Bacterianos/inmunología , Western Blotting , Proliferación Celular , Inmunidad Celular , Inmunidad Humoral , Linfocitos/citología , Ratones , Plásmidos , Proteínas Recombinantes de Fusión/inmunología , Bazo/citologíaRESUMEN
Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Perfilación de la Expresión Génica , Muromegalovirus/inmunología , Latencia del Virus , Animales , Antígenos Virales/biosíntesis , Modelos Animales de Enfermedad , Epítopos/biosíntesis , Epítopos/inmunología , Memoria Inmunológica , Muromegalovirus/crecimiento & desarrolloRESUMEN
To enhance the synthesis of antigenic envelope proteins L1 of high-grade papillomavirus types HPV16, HPV18, HPV31, and HPV45, the sequence of the gene encoding the cucumber mosaic virus replicase (RdRP CMV) was inserted into the genetic construct. This made it possible to increase the production of these antigenic proteins to 25-27 µg/mg total soluble protein.
Asunto(s)
Cucumovirus , Epítopos , Papillomaviridae/genética , Vacunas contra Papillomavirus , Solanum lycopersicum , Proteínas Virales , Cucumovirus/genética , Cucumovirus/metabolismo , Epítopos/biosíntesis , Epítopos/química , Epítopos/genética , Solanum lycopersicum/química , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/virología , Vacunas contra Papillomavirus/biosíntesis , Vacunas contra Papillomavirus/química , Vacunas contra Papillomavirus/genética , Proteínas Virales/biosíntesis , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Sarcocystidae/inmunología , Animales , Biomarcadores , Western Blotting , Electroforesis en Gel de Poliacrilamida , Epítopos/biosíntesis , Epítopos/inmunología , Hibridomas/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Electrónica de Transmisión , Sarcocystidae/ultraestructuraRESUMEN
Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing cells was confirmed by flow cytometry. The fusion protein induced a strong signal transduction on human monocytes via FcγRI. The expression vector pTriEx-3 Neo containing the described construct was used as a naked DNA vaccine and introduced directly to experimental humanized NOD SCID gamma mice with or without boosting with the expressed fusion protein. Immunization with the generated DNA chimeric molecules and prime-boost with the expressed recombinant proteins induced significant serum levels of anti-influenza immunoglobulin G antibodies and strong cytotoxic T lymphocyte activity against influenza virus-infected cells in humanized animals.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Células Presentadoras de Antígenos/inmunología , Células CHO , Cricetulus , Epítopos/biosíntesis , Citometría de Flujo , Regulación de la Expresión Génica , Ingeniería Genética , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Orthomyxoviridae/inmunología , Orthomyxoviridae/patogenicidad , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg-1 at 90% purity. Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL-1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution. We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Química Farmacéutica/métodos , Epítopos/química , Animales , Anticuerpos Monoclonales/metabolismo , Cromatografía de Afinidad , Epítopos/biosíntesis , Epítopos/metabolismo , Anticuerpos Anti-VIH/metabolismo , Ligandos , Proteínas Luminiscentes/química , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Aberrant glycan epitopes are a classic hallmark of malignant transformation, yet their full clinical potential in cancer diagnostics and therapeutics is yet to be realized. This is partly because our understanding of how these epitopes are regulated remains poorly understood. In this review cancer glycan epitopes for the major glycan classes are summarized with a focus on their biosynthesis, structure and role in cancer progression. Their application as cancer biomarkers, in particular the more recent work on cancer glycoforms, and the advantages these offer over the glycan or protein alone are discussed. Finally, emerging concepts which expand on the current view of the cancer glycan epitope beyond the single structure, to patterns and the whole glycocalyx, are described. These new approaches that consider the cancer glycan epitope as a glycoform, or as a pattern of many epitope structures, are providing new targets both for cancer biomarkers and therapeutics currently in development at the bench and the clinic.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Epítopos/química , Epítopos/metabolismo , Neoplasias/química , Neoplasias/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/química , Epítopos/biosíntesis , Humanos , Polisacáridos/biosíntesisRESUMEN
Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization.
Asunto(s)
Adenosina Trifosfatasas , Anticuerpos/química , Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte de Catión , Moléculas de Adhesión Celular Neuronal , Epítopos , Regulación de la Expresión Génica , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/química , Animales , Astrocitos/patología , Encéfalo/patología , Lesiones Encefálicas/patología , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/química , Moléculas de Adhesión Celular Neuronal/biosíntesis , Moléculas de Adhesión Celular Neuronal/química , Modelos Animales de Enfermedad , Epítopos/biosíntesis , Epítopos/química , Femenino , Masculino , RatonesRESUMEN
Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of ß-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections.
Asunto(s)
Anticuerpos Antivirales , Antígenos Virales , Virus del Dengue/genética , Dengue , Epítopos , Inmunoglobulina M , Proteínas Virales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/aislamiento & purificación , Dengue/sangre , Dengue/diagnóstico , Dengue/inmunología , Virus del Dengue/inmunología , Virus del Dengue/metabolismo , Epítopos/biosíntesis , Epítopos/genética , Epítopos/inmunología , Epítopos/aislamiento & purificación , Humanos , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/aislamiento & purificaciónRESUMEN
Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487LeX, 5750LeX and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage, 487LeX-, 5750LeX- and 473HD-related glycans were differently expressed. Later, cells of the three germ layers in embryoid bodies (hEBs) and, even after neuralization of hEBs, subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC), LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCsFGF-2/EGF derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCsFGF-2/EGF. Finally, we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487LeX, 5750LeX and 473HD are promising tools for identifying distinct stages during neural differentiation.
Asunto(s)
Diferenciación Celular , Glicoconjugados/fisiología , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/biosíntesis , Línea Celular , Polaridad Celular , Epítopos/biosíntesis , Glicoconjugados/biosíntesis , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/inmunología , Polisacáridos/biosíntesisRESUMEN
After more than a decade of biomarker discovery using advanced proteomic and genomic approaches, very few biomarkers have been involved in clinical diagnostics. Most candidate biomarkers are focused on the protein component. Targeting post-translational modifications (PTMs) in combination with protein sequences will provide superior diagnostic information with regards to sensitivity and specificity. Glycosylation is one of the most common and functionally important PTMs. It plays a central role in many biological processes, including protein folding, host-pathogen interactions, immune response, and inflammation. Cancer-associated aberrant glycosylation has been identified in various types of cancer. Expression of cancer-specific glycan epitopes represents an excellent opportunity for diagnostics and potentially specific detection of tumors. Here, we report four proteins (LIFR, CE350, VP13A, HPT) found in sera from pancreatic cancer patients carrying aberrant glycan structures as compared to those of controls.
Asunto(s)
Biomarcadores de Tumor/sangre , Haptoglobinas/análisis , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/sangre , Proteínas de Microtúbulos/sangre , Proteínas Nucleares/sangre , Neoplasias Pancreáticas/sangre , Proteínas de Transporte Vesicular/sangre , Anciano , Epítopos/biosíntesis , Epítopos/química , Epítopos/genética , Femenino , Glicosilación , Interacciones Huésped-Patógeno/genética , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Polisacáridos/biosíntesis , Polisacáridos/química , Polisacáridos/genética , Pliegue de Proteína , Procesamiento Proteico-Postraduccional/genética , ProteómicaRESUMEN
Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus-like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self-assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N- and C-termini and a predicted antigenic region located near the N-terminus. Wild-type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope-presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small-scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2.
Asunto(s)
Proteínas de la Cápside , Circovirus , Epítopos , Expresión Génica , Virus de Plantas , Vacunas Virales , Animales , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Circovirus/genética , Circovirus/inmunología , Epítopos/biosíntesis , Epítopos/genética , Epítopos/inmunología , Ratones , Virus de Plantas/genética , Virus de Plantas/inmunología , Virus de Plantas/metabolismo , Síndrome Multisistémico de Emaciación Posdestete Porcino/inmunología , Síndrome Multisistémico de Emaciación Posdestete Porcino/prevención & control , Porcinos , Vacunas Virales/biosíntesis , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Olive seeds, which are a raw material of interest, have been reported to contain 11S seed storage proteins (SSPs). However, the presence of SSPs such as 7S vicilins has not been studied. In this study, following a search in the olive seed transcriptome, 58 sequences corresponding to 7S vicilins were retrieved. A partial sequence was amplified by PCR from olive seed cDNA and subjected to phylogenetic analysis with other sequences. Structural analysis showed that olive 7S vicilin contains 9 α-helixes and 22 ß-sheets. Additionally, 3D structural analysis displayed good superimposition with vicilin models generated from Pistacia and Sesamum. In order to assess potential allergenicity, T and B epitopes present in these proteins were identified by bioinformatic approaches. Different motifs were observed among the species, as well as some species-specific motifs. Finally, expression analysis of vicilins was carried out in protein extracts obtained from seeds of different species, including the olive. Noticeable bands were observed for all species in the 15-75 kDa MW interval, which were compatible with vicilins. The reactivity of the extracts to sera from patients allergic to nuts was also analysed. The findings with regard to the potential use of olive seed as food are discussed.