RESUMEN
Mucosal immunity plays a crucial role in combating and controlling the spread of highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recombinant subunit vaccines have shown safety and efficacy in clinical trials, but further investigation is necessary to evaluate their feasibility as mucosal vaccines. This study developed a SARS-CoV-2 mucosal vaccine using spike (S) proteins from a prototype strain and the omicron variant, along with a cationic chitosan adjuvant, and systematically evaluated its immunogenicity after both primary and booster immunization in mice. Primary immunization through intraperitoneal and intranasal administration of the S protein elicited cross-reactive antibodies against prototype strains, as well as delta and omicron variants, with particularly strong effects observed after mucosal vaccination. In the context of booster immunization following primary immunization with inactivated vaccines, the omicron-based S protein mucosal vaccine resulted in a broader and more robust neutralizing antibody response in both serum and respiratory mucosa compared to the prototype vaccine, enhancing protection against different variants. These findings indicate that mucosal vaccination with the S protein has the potential to trigger a broader and stronger antibody response during primary and booster immunization, making it a promising strategy against respiratory pathogens.
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Administración Intranasal , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Inmunización Secundaria , Ratones Endogámicos BALB C , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Ratones , Inmunización Secundaria/métodos , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , SARS-CoV-2/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Femenino , Inmunidad Mucosa , Inmunogenicidad Vacunal , Reacciones Cruzadas/inmunología , Quitosano/inmunología , Quitosano/administración & dosificación , Adyuvantes de Vacunas/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/administración & dosificaciónRESUMEN
The influenza A virus (IAV) is a ubiquitous and continuously evolving respiratory pathogen. The intranasal vaccination mimicking natural infections is an attractive strategy for controlling IAVs. Multiepitope vaccines accurately targeting multiple conserved domains have the potential to broaden the protective scope of current seasonal influenza vaccines and reduce the risk of generating escape mutants. Here, multiple linear epitopes from the matrix protein 2 ectodomain (M2e) and the hemagglutinin stem domain (HA2) are fused with the Helicobacter pylori ferritin, a self-assembled nanocarrier and mucosal adjuvant, to develop a multiepitope nanovaccine. Through intranasal delivery, the prokaryotically expressed multiepitope nanovaccine elicits long-lasting mucosal immunity, broad humoral immunity, and robust cellular immunity without any adjuvants, and confers complete protection against H3N2 and H1N1 subtypes of IAV in mice. Importantly, this intranasal multiepitope nanovaccine triggers memory B-cell responses, resulting in secretory immunoglobulin A (sIgA) and serum immunoglobulin G (IgG) levels persisting for more than five months post-immunization. Therefore, this intranasal ferritin-based multiepitope nanovaccine represents a promising approach to combating respiratory pathogens.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae , Animales , Ratones , Infecciones por Orthomyxoviridae/prevención & control , Subtipo H3N2 del Virus de la Influenza A , Nanovacunas , Inmunidad Mucosa , Ferritinas , Anticuerpos Antivirales , Adyuvantes Inmunológicos/farmacología , Administración Intranasal , Adyuvantes Farmacéuticos , Ratones Endogámicos BALB CRESUMEN
Seasonal influenza vaccines typically provide strain-specific protection and are reformulated annually, which is a complex and time-consuming process. Multiepitope vaccines, combining multiple conserved antigenic epitopes from a pathogen, can trigger more robust, diverse, and effective immune responses, providing a potential solution. However, their practical application is hindered by low immunogenicity and short-term effectiveness. In this study, multiple linear epitopes from the conserved stem domain of hemagglutinin and the ectodomain of matrix protein 2 are combined with the Helicobacter pylori ferritin, a stable self-assembled nanoplatform, to develop an influenza multiepitope nanovaccine, named MHF. MHF is prokaryotically expressed in a soluble form and self-assembles into uniform nanoparticles. The subcutaneous immunization of mice with adjuvanted MHF induces cross-reactive neutralizing antibodies, antibody-dependent cell-mediated cytotoxicity, and cellular immunity, offering complete protection against H3N2 as well as partial protection against H1N1. Importantly, the vaccine cargo delivered by ferritin triggers epitope-specific memory B-cell responses, with antibody level persisting for over 6 months post-immunization. These findings indicate that self-assembled multiepitope nanovaccines elicit potent and long-lasting immune responses while significantly reducing the risk of vaccine escape mutants, and offer greater practicality in terms of scalable manufacturing and genetic manipulability, presenting a promising and effective strategy for future vaccine development.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Infecciones por Orthomyxoviridae , Animales , Ratones , Humanos , Nanovacunas , Subtipo H3N2 del Virus de la Influenza A , Anticuerpos Antivirales , Epítopos , Ferritinas/metabolismo , Ratones Endogámicos BALB CRESUMEN
Introduction: Broadly neutralizing antibodies (bNAbs) have the ability to neutralize a considerable breadth of genetically diverse human immunodeficiency virus (HIV) strains. Passive immunization can potentially provide protection against HIV infection in animal models. However, the direct antibody infusion effect is limited due to the short half-life and deficient immunogenicity of the antibody. As an alternative strategy, we propose the use of nano viral vectors, specifically the adeno-associated virus (AAV), to continuously and systematically produce bNAbs against HIV. Methods: Plasmids expressing bNAbs PG9, PG16, 10E8, and NIH45-46 antibodies were constructed, targeting three different epitopes of HIV. Additionally, the bNAbs gene mediated by rAAV8 was administered to generate long-term expression with a single injection. We established both single and combined immunization groups. The neutralizing activity of antibodies expressed in mice sera was subsequently evaluated. Results: The expression of bNAbs in BALB/c mice can last for >24 weeks after a single intramuscular injection of rAAV8. Further studies show that neutralization of the HIV pseudovirus by sera from co-immunized mice with rAAV8 expressing 10E8 and PG16 was enhanced compared with mice immunized with 10E8 or PG16 alone. Conclusion: The prolonged expression of neutralizing antibodies can be maintained over long periods in BALB/c mice. This combined immunization is a promising candidate strategy for HIV treatment.
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Infecciones por VIH , VIH-1 , Humanos , Animales , Ratones , Infecciones por VIH/prevención & control , VIH-1/genética , Anticuerpos ampliamente neutralizantes , Anticuerpos Neutralizantes , Inmunización , Ratones Endogámicos BALB CRESUMEN
Polysaccharides are one of the most abundant and active components of Lysimachia christinae (L. christinae), which is widely adopted for attenuating abnormal cholesterol metabolism; however, its mechanism of action remains unclear. Therefore, we fed a natural polysaccharide (NP) purified from L. christinae to high-fat diet mice. These mice showed an altered gut microbiota and bile acid pool, which was characterized by significantly increased Lactobacillus murinus and unconjugated bile acids in the ileum. Oral administration of the NP reduced cholesterol and triglyceride levels and enhanced bile acid synthesis via cholesterol 7α-hydroxylase. Additionally, the effects of NP are microbiota-dependent, which was reconfirmed by fecal microbiota transplantation (FMT). Altered gut microbiota reshaped bile acid metabolism by modulating bile salt hydrolase (BSH) activity. Therefore, bsh genes were genetically engineered into Brevibacillus choshinensis, which was gavaged into mice to verify BSH function in vivo. Finally, adeno-associated-virus-2-mediated overexpression or inhibition of fibroblast growth factor 15 (FGF15) was used to explore the farnesoid X receptor-fibroblast growth factor 15 pathway in hyperlipidemic mice. We identified that the NP relieves hyperlipidemia by altering the gut microbiota, which is accompanied by the active conversion of cholesterol to bile acids.
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Microbioma Gastrointestinal , Hiperlipidemias , Ratones , Animales , Lysimachia , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/etiología , Hiperlipidemias/metabolismo , Ácidos y Sales Biliares/metabolismo , Polisacáridos/farmacología , Polisacáridos/metabolismo , Colesterol/metabolismo , Dieta Alta en Grasa/efectos adversos , Transducción de Señal , Factores de Crecimiento de Fibroblastos/metabolismo , Ratones Endogámicos C57BL , HígadoRESUMEN
Current seasonal influenza vaccines confer only limited coverage of virus strains due to the frequent genetic and antigenic variability of influenza virus (IV). Epitope vaccines that accurately target conserved domains provide a promising approach to increase the breadth of protection; however, poor immunogenicity greatly hinders their application. The protruding (P) domain of the norovirus (NoV), which can self-assemble into a 24-mer particle called the NoV P particle, offers an ideal antigen presentation platform. In this study, a multiepitope nanovaccine displaying influenza epitopes (HMN-PP) was constructed based on the NoV P particle nanoplatform. Large amounts of HMN-PP were easily expressed in Escherichia coli in soluble form. Animal experiments showed that the adjuvanted HMN-PP nanovaccine induced epitope-specific antibodies and haemagglutinin (HA)-specific neutralizing antibodies, and the antibodies could persist for at least three months after the last immunization. Furthermore, HMN-PP induced matrix protein 2 extracellular domain (M2e)-specific antibody-dependent cell-mediated cytotoxicity, CD4+ and CD8+ T-cell responses, and a nucleoprotein (NP)-specific cytotoxic T lymphocyte (CTL) response. These results indicated that the combination of a multiepitope vaccine and self-assembled NoV P particles may be an ideal and effective vaccine strategy for highly variable viruses such as IV and SARS-CoV-2. Electronic Supplementary Material: Supplementary material is available in the online version of this article at 10.1007/s12274-023-5395-6.
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We described an operation that co-overexpress interleukin receptor 1 (IL-1R1) and its co-receptor (IL-1R1AcP) genes in wild-type A375·S2 cells in order to increase their sensibility to IL-1. Firstly, laser scanning confocal microscope observed that IL-1R1 could be expressed on the surface of A375·S2 cells. qPCR was performed to estimate the ratio of two genes and result showed the ratio was almost 4.57:1. Then two genes were linked to vectors and co-transfected into A375·S2 cells. qPCR and Western blotting showed the protein content improved markedly. Finally, MTS assay was executed and the sensitivity of A375·S2 cells that co-transfected receptors to IL-1ß increased significantly. Another MTS assay showed the cell activity variation changed significantly (P < 0.05) and the reliability of the experiment was high, indicating that cell line established in this study could be further used for the activity test of IL-1Ra. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01027-8.
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COVID-19 is an emerging disease that poses a severe threat to global public health. As such, there is an urgent demand for vaccines against SARS-CoV-2, the virus that causes COVID-19. Here, we describe a virus-like nanoparticle candidate vaccine against SARS-CoV-2 produced by an E. coli expression system. The fusion protein of a truncated ORF2-encoded protein of aa 439~608 (p170) from hepatitis E virus CCJD-517 and the receptor-binding domain of the spike protein from SARS-CoV-2 were expressed, purified and characterized. The antigenicity and immunogenicity of p170-RBD were evaluated in vitro and in Kunming mice. Our investigation revealed that p170-RBD self-assembled into approximately 24 nm virus-like particles, which could bind to serum from vaccinated people (p < 0.001) and receptors on cells. Immunization with p170-RBD induced the titer of IgG antibody vaccine increased from 14 days post-immunization and was significantly enhanced after a booster immunization at 28 dpi, ultimately reaching a peak level on 42 dpi with a titer of 4.97 log10. Pseudovirus neutralization tests showed that the candidate vaccine induced a strong neutralizing antibody response in mice. In this research, we demonstrated that p170-RBD possesses strong antigenicity and immunogenicity and could be a potential candidate for use in future SARS-CoV-2 vaccine development.
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COVID-19 , Virus de la Hepatitis E , Vacunas Virales , Animales , Humanos , Ratones , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Proteínas de la Cápside/genética , COVID-19/prevención & control , Vacunas contra la COVID-19/genética , Escherichia coli , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Vacunas Virales/genéticaRESUMEN
BACKGROUND: Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. RESULT: The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. CONCLUSIONS: YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.
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Virus del Moquillo Canino , Ferritinas/química , Hemaglutininas Virales , Nanopartículas del Metal/química , Vacunas de ADN , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Citocinas/metabolismo , Moquillo/prevención & control , Virus del Moquillo Canino/química , Virus del Moquillo Canino/inmunología , Perros , Femenino , Células HEK293 , Hemaglutininas Virales/química , Hemaglutininas Virales/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Vacunas de ADN/química , Vacunas de ADN/inmunología , Células VeroRESUMEN
Influenza viruses continue to threaten public health, and currently available vaccines provide insufficient immunity against seasonal and pandemic influenza. The use of recombinant trimeric hemagglutinin (HA) as an Ag provides an attractive alternative to current influenza vaccines. Aiming to develop an effective vaccine with rapid production, robust immunogenicity, and high protective efficiency, a DNA vaccine was designed by fusing influenza virus HA with self-assembled ferritin nanoparticles, denoted as HA-F. This candidate vaccine was prepared and purified in a 293-6E cell eukaryotic expression system. After BALB/c mice were immunized with 100 µg of HA-F DNA 3 times, HA-F elicited significant HA-specific humoral immunity and T cell immune responses. The HA-F DNA vaccine also conferred protection in mice against a lethal infection of homologous A/17/California/2009/38 (H1N1) virus. These results suggest that the HA-F DNA vaccine is a competitive vaccine candidate and presents a promising vaccination approach against influenza viruses.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Nanopartículas , Infecciones por Orthomyxoviridae , Vacunas de ADN , Animales , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Hemaglutininas , Humanos , Gripe Humana/prevención & control , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Influenza A viruses (IAVs) induce acute respiratory disease and cause severe epidemics and pandemics. Since IAVs exhibit antigenic variation and genome reassortment, the development of broad-spectrum influenza vaccines is crucial. The stem of the hemagglutinin (HA) is highly conserved across IAV strains and thus has been explored in broad-spectrum influenza vaccine studies. The present study aimed to identify viral epitopes capable of eliciting effective host immune responses, which can be explored for the development of broad-spectrum non-strain specific prophylactic options against IAV. METHODS: In this study, a series of conserved linear sequences from the HA stem of IAV (H1N1) was recognized by sequence alignment and B/T-cell epitope prediction after being chemically coupled to the Keyhole Limpet Hemocyanin (KLH) protein. The predicted linear epitopes were identified by enzyme-linked immunosorbent assay (ELISA) after animal immunization and then fused with ferritin carriers. RESULTS: Three predicted linear epitopes with relatively strong immunogenicity, P3, P6 and P8 were fused with ferritin carriers P3F, P6F and P8F, respectively to further improve their immunogenicity. Antibody titre of the sera of mice immunized with the recombinant immunogens revealed the elicitation of specific antibody-binding activities by the identified sequences. While hemagglutinin-inhibition activities were not detected in the antisera, neutralizing antibodies against the H1 and H3 virus subtypes were detected by the microneutralization assay. CONCLUSION: The linear epitopes fused with ferritin identified in this study can lay the foundation for future advancements in development of broad-spectrum subunit vaccine against IAV (H1N1), and give rise to the potential future applicability of ferritin-based antigen delivery nanoplatforms.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Infecciones por Orthomyxoviridae , Animales , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Hemaglutininas/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/prevención & control , Ratones , Infecciones por Orthomyxoviridae/prevención & control , Péptidos/genéticaRESUMEN
BACKGROUND: Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide. OBJECTIVES: A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy. METHODS: In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer. RESULTS: The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2. CONCLUSIONS: The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.
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Proteínas de la Cápside/genética , Circovirus/inmunología , Dependovirus/genética , Inmunidad Celular , Inmunidad Humoral , Vacunas Virales/inmunología , Animales , Proteínas de la Cápside/metabolismo , Circovirus/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
Copper is an essential element in living systems and plays an important role in human physiology; therefore, methods to detect the concentration of copper ions in living organisms are important. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe that can be used for the detection of Cu2+. The probe, BNQ, has high selectivity and sensitivity. The fluorescence intensity of the probe at 520 nm was visible to the naked eye under a UV lamp; upon the gradual addition of Cu2+, there was a colour change from green to nearly colourless. Furthermore, the detection limit of BNQ for Cu2+ was 45.5 nM. The detection mechanism was investigated using a Job's plot and density functional theory (DFT) calculations. In addition, owing to great biocompatibility, we were able to successfully use BNQ to detect Cu2+ in living HeLa cells with low toxicity.
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A fluorescent probe that responds at distinct wavelengths upon exposure to cyanide, hypochlorite, and bisulfite was synthesized. As a result, an easy to apply analytical methodology was developed for the detection of these ions. The feasibility of this method was evaluated by theoretical calculations. The probe exhibited excellent solubility in the test solution (H2O: DMF = 99: 1, v: v) with low detection limits for cyanide, hypochlorite and bisulfite (4.5 × 10 -8 M, 4.9 × 10 -7 M and 4.3 × 10 -8 M respectively) showing distinct emission wavelengths for each ion without interference in practical application. Furthermore, the probe had low toxicity and was applied for the imaging experiments of cyanide, hypochlorite and bisulfite in living HeLa and MDCK cells.
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Cianuros/análisis , Colorantes Fluorescentes , Ácido Hipocloroso/análisis , Imagen Óptica , Sulfitos/análisis , Agua/química , Animales , Perros , Células HeLa , Humanos , Límite de Detección , Células de Riñón Canino Madin DarbyRESUMEN
Alzheimer's disease (AD) is the most prevalent form of dementia worldwide and is an emerging global epidemic. Active and passive immune therapies targeting beta amyloid (Aß) have shown very limited evidence in human studies of clinical benefits from these approaches. Epidemiological studies have shown that subjects with type 2 diabetes (T2D) are at higher risk of developing AD. However, whether and how these two conditions are causally linked is unknown. With the purpose of confirming the relationship between T2D and AD, this study specifically focused on effects of insulin in an in vitro model of the human blood-brain barrier (BBB) and on potential mechanisms of action in the treatment of AD. By using a series of assays to establish a BBB model, we demonstrated that insulin treatment alone could induce the increase of brain endothelial barrier properties. The transcriptional response of hCMEC/D3 cells to activation with different concentrations of insulin was determined by RT-PCR, and expression levels of genes involved in the control of barrier permeability, including inter-brain endothelial junctions, integrin-focal adhesions complexes, and transporter system, were found to be altered by the treatment. Notably, the influence of insulin on expression of the ATP-binding cassette (ABC) transporter which contributes to the clearance of Aß was investigated. Insulin up-regulated adherens junction and tight junction transmembrane proteins, as well as the ABC transporter. By treatment with insulin, the models have major advantages: it is fast, it has low cost, it is fit for considerable samples, and its conditions are under control.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Endotelio Vascular/metabolismo , Insulina/farmacología , Transcriptoma/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Péptidos beta-Amiloides/metabolismo , Transporte Biológico , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/citología , Encéfalo/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Hipoglucemiantes/farmacología , Técnicas In Vitro , Modelos BiológicosRESUMEN
According to the binding site structure and the catalytic mechanism of the native glutathione peroxidase (GPX), three glutathione derivatives, GSH-S-DNP butyl ester (hapten Be), GSH-S-DNP hexyl ester (hapten He) and GSH-S-DNP hexamethylene ester (hapten Hme) were synthesized. By a four-round panning with a human synthetic scFv phage library against three haptens, the enrichment of the scFv phage particles with specific binding activity could be determined. Three phage particles were selected binding to each glutathione derivative, respectively. After a two-step chemical mutation to convert the serine residues of the scFv phage particles into selenocysteine residues, GPX activity could be observed and determined upto 3000 U µmol-1 in the selenium-containing scFv phage abzyme which was isolated by affinity capture against the hapten Be. Also the scFv phage abzymes elicited by different antigens displayed different catalytic activities. After a directed evolution by DNA shuffling to improve the affinity to the hapten Be, a secondary library with GPX activity was created in which the catalytic activity of the selenium-containing scFv phage abzyme could be increased 17%. This study might be helpful for new haptens or antigens design to optimize the abzymes with high binding activities and might also provide a novel scheme for GPX mimic candidates for drug development.