RESUMO
Adaptive laboratory evolution has been used to improve production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) in insect cells. However, little is known about the underlying biological mechanisms promoting higher HA-VLP expression in such adapted cell lines. In this article, we present a study of gene expression patterns associated with high-producer insect High Five cells adapted to neutral pH, in comparison to non-adapted cells, during expression of influenza HA-VLPs. RNA-seq shows a decrease in the amount of reads mapping to host cell genomes along infection, and an increase in those mapping to baculovirus and transgenes. A total of 1742 host cell genes were found differentially expressed between adapted and non-adapted cells throughout infection, 474 of those being either up- or down-regulated at both time points evaluated (12 and 24 h post-infection). Interestingly, while host cell genes were found up- and down-regulated in an approximately 1:1 ratio, all differentially expressed baculovirus genes were found to be down-regulated in infected adapted cells. Pathway analysis of differentially expressed genes revealed enrichment of ribosome biosynthesis and carbohydrate, amino acid, and lipid metabolism. In addition, oxidative phosphorylation and protein folding, sorting and degradation pathways were also found to be overrepresented. These findings contribute to our knowledge of biological mechanisms of insect cells during baculovirus-mediated transient expression and will assist the identification of potential engineering targets to increase recombinant protein production in the future.
Assuntos
Vacinas contra Influenza , Influenza Humana , Animais , Humanos , Hemaglutininas/genética , Baculoviridae/genética , Insetos/genética , Proteínas Recombinantes/genética , Expressão Gênica , Aminoácidos/genética , Carboidratos , RNARESUMO
Abstract Baculoviruses are considered as effective bio pesticides except of being not active under sunlight conditions. The aim of this study is to evaluate the capability of moringa extract to prolong virus activity under Egyptian field conditions especially that Moringa proved to be strong protective material under previous investigation under laboratory conditions the addition of moringa filters were tested on tomato plant foliage. Results are based on leaf bioassay using Spodoptera littoralis test insect and its nuclepolyhedrovirus (SpliNPV) as standard materials. The Original Activity Remaining (OAR) and Lethal Infectivity Time to 50% (LIT50) were estimated after exposure to natural sunlight. cacao and green tea were tested as comparative materials, which proved to be effective as virus protective agent in earlier investigations. The results showed that moringa additive at 10% sustained 50% of virus activity for 193.53 hours and 62.05 and 23.023 hours post application for cacao and green tea; respectively. While virus alone treatment lasts for only 17.551 hours. Moringa generally available, relatively cheap; it also has been tested and proved to be non-toxic, safe, and friendly to the environment. The obtained results showed the activity of moringa water extract in prolonging the virus activity under field application.
Resumo Os baculovírus são considerados como biopesticidas eficazes, exceto por não estarem ativos sob condições de luz solar. O objetivo deste estudo é avaliar a capacidade do extrato de moringa para prolongar a atividade do vírus sob condições de campo egípcias, tendo em vista que Moringa provou ser um material protetor forte sob investigação anterior em condições de laboratório a adição de filtros moringa foram testados na folhagem de plantas de tomate. Os resultados são com base em bioensaios foliares utilizando o inseto-teste Spodoptera littoralis e seu vírus de poliedrose nuclear (VPNSl) como materiais padrões. A Atividade Original Restante (AOR) e o Tempo de Infectividade Letal até 50% (LIT50) foram estimados após a exposição à luz solar natural. Cacau e chá verde foram testados como materiais comparativos, que se mostraram eficazes como agentes protetores do vírus em investigações anteriores. Os resultados mostraram que a moringa aditiva a 10% sustentou 50% da atividade viral por 193,53 horas e 62,05 e 23,023 horas após a aplicação de cacau e chá verde, respectivamente. Enquanto o tratamento sozinho do vírus dura apenas 17,551 horas, a moringa geralmente está disponível, e é relativamente barata; e a mesma também foi testada e provou ser não tóxica, segura e propícia ao meio ambiente. Os resultados obtidos mostraram a atividade do extrato aquosa da moringa no prolongamento da atividade do vírus sob aplicação em campo.
Assuntos
Animais , Moringa , Luz Solar , Água , Extratos Vegetais/farmacologia , Baculoviridae , Folhas de Planta , EgitoRESUMO
Baculoviruses are considered as effective bio pesticides except of being not active under sunlight conditions. The aim of this study is to evaluate the capability of moringa extract to prolong virus activity under Egyptian field conditions especially that Moringa proved to be strong protective material under previous investigation under laboratory conditions the addition of moringa filters were tested on tomato plant foliage. Results are based on leaf bioassay using Spodoptera littoralis test insect and its nuclepolyhedrovirus (SpliNPV) as standard materials. The Original Activity Remaining (OAR) and Lethal Infectivity Time to 50% (LIT50) were estimated after exposure to natural sunlight. cacao and green tea were tested as comparative materials, which proved to be effective as virus protective agent in earlier investigations. The results showed that moringa additive at 10% sustained 50% of virus activity for 193.53 hours and 62.05 and 23.023 hours post application for cacao and green tea; respectively. While virus alone treatment lasts for only 17.551 hours. Moringa generally available, relatively cheap; it also has been tested and proved to be non-toxic, safe, and friendly to the environment. The obtained results showed the activity of moringa water extract in prolonging the virus activity under field application.
Assuntos
Moringa , Animais , Baculoviridae , Egito , Extratos Vegetais/farmacologia , Folhas de Planta , Luz Solar , ÁguaRESUMO
The baculovirus-insect cell expression system has been widely used for heterologous protein expression and virus-like particles (VLPs) expression. In this study, we established a new method for antiviral screening targeting to glycoprotein E of flaviviruses based on the baculovirus expression system. ZIKV is a mosquito-borne flavivirus and has posed great threat to the public health. It has been reported that ZIKV infection was associated with microcephaly and serious neurological complications. Our study showed that either ZIKV E or prME protein expressed in insect cells can form VLPs and induce membrane fusion between insect cells. Therefore, the E protein, which is responsible for receptor binding, attachment, and virus fusion during viral entry, achieved proper folding and retained its fusogenic ability in VLPs when expressed in this system. The syncytia in insect cells were significantly reduced by the anti-ZIKV-E specific polyclonal antibody in a dose-dependent manner. AMS, a thiol-conjugating reagent, was also shown to have an inhibitory effect on the E protein induced syncytia and inhibited ZIKV infection by blocking viral entry. Indeed the phenomenon of syncytial formation induced by E protein expressed VLPs in insect cells is common among flaviviruses, including Japanese encephalitis virus (JEV), Dengue virus type 2 (DENV-2), and tick-borne encephalitis virus (TBEV). This inhibition effect on syncytial formation can be developed as a novel, safe, and simple antiviral screening approach for inhibitory antibodies, peptides, or small molecules targeting to E protein of ZIKV and other flaviviruses.
Assuntos
Baculoviridae/genética , Infecções por Flavivirus/virologia , Flavivirus/fisiologia , Expressão Gênica , Vetores Genéticos/genética , Células Gigantes/virologia , Animais , Antivirais/farmacologia , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Flavivirus/efeitos dos fármacos , Flavivirus/ultraestrutura , Engenharia Genética , Humanos , Transporte Proteico , Ensaio de Placa Viral , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment.
Assuntos
Detergentes/química , Ligantes , Micelas , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Acridinas/química , Trifosfato de Adenosina/química , Animais , Baculoviridae/metabolismo , Sítios de Ligação , Dibenzocicloeptenos/química , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Glucosídeos/química , Humanos , Hidrólise , Concentração Inibidora 50 , Insetos , Camundongos , Peptídeos Cíclicos/química , Ligação Proteica , Quinolinas/química , Tetra-Hidroisoquinolinas/química , Verapamil/químicaRESUMO
α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72-96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP.
Assuntos
Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , alfa-Fetoproteínas/biossíntese , alfa-Fetoproteínas/farmacologia , Animais , Baculoviridae , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Fatores Imunológicos/biossíntese , Fatores Imunológicos/química , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Insetos/citologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , alfa-Fetoproteínas/química , alfa-Fetoproteínas/isolamento & purificaçãoRESUMO
There is an urgent need for Ebola virus (EBOV) proteins, EBOV-specific antibodies and recombinant antigens to be used in diagnostics and as potential vaccine candidates. Our objective was to produce and purify recombinant proteins for immunological assays and for the production of polyclonal EBOV specific antibodies. In addition, a limited comparison of the adjuvant effects of Freund's complete adjuvant (FCA) and adjuvant system 03 (AS03) was carried out. Recombinant EBOV GST-VP24, -VP30, -VP35, -VP40 and -NP were produced in E. coli and purified with affinity chromatography followed by preparative gel electrophoresis. Recombinant EBOV GP-His was produced in Sf9 insect cells and purified by preparative gel electrophoresis. To compare the adjuvant effect of FCA and AS03, 12 rabbits were immunized four times with one of the six recombinant EBOV proteins using FCA or AS03. In addition, three guinea pigs were immunized with EBOV VP24 using FCA. With the exception of sera from two rabbits immunized with GST-VP24, the antisera against all other EBOV proteins showed very high and specific antibody responses after three to four immunizations. The adjuvant effect of AS03 was comparable to that of FCA. The produced antibodies recognized the corresponding EBOV proteins in wild type EBOV-infected cells.
Assuntos
Adjuvantes Imunológicos , Ebolavirus/imunologia , Adjuvante de Freund , Polissorbatos , Esqualeno , Proteínas Virais/imunologia , Proteínas Virais/isolamento & purificação , alfa-Tocoferol , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Baculoviridae/genética , Combinação de Medicamentos , Ebolavirus/química , Ebolavirus/isolamento & purificação , Adjuvante de Freund/provisão & distribuição , Cobaias , Coelhos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/genéticaRESUMO
Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever and has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. A bicistronic baculovirus expression system was utilized to co-express CHIKV structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium is mediated by the CHIKV E1 allowing it to identify chemicals that can prevent syncytium formation. The compounds characterized by this method could be anti-CHIKV drugs.
Assuntos
Antivirais/farmacologia , Baculoviridae/genética , Proteínas do Capsídeo/genética , Vírus Chikungunya/efeitos dos fármacos , Proteínas do Envelope Viral/genética , Animais , Baculoviridae/metabolismo , Proteínas do Capsídeo/metabolismo , Fusão Celular , Vírus Chikungunya/genética , Avaliação Pré-Clínica de Medicamentos , Vetores Genéticos/genética , Células Gigantes/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , Células Sf9 , Proteínas do Envelope Viral/metabolismoRESUMO
Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Baculoviridae/genética , Proliferação de Células , China , Peste Suína Clássica/imunologia , Peste Suína Clássica/patologia , Vírus da Febre Suína Clássica/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos , Injeções Intramusculares , Linfócitos/imunologia , Testes de Neutralização , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificaçãoRESUMO
Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.
Assuntos
Aminoácidos/química , Antitrombinas/química , Fator IXa/química , Inibidores do Fator Xa/química , Fator Xa/química , Heparina/química , Regulação Alostérica , Aminoácidos/metabolismo , Antitrombinas/metabolismo , Baculoviridae/genética , Sítios de Ligação , Fator IXa/genética , Fator IXa/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Inibidores do Fator Xa/metabolismo , Expressão Gênica , Heparina/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
ß-Adrenergic agonists (ß-agonists) are illegally used in animal husbandry, threatening the health of consumers. To realize multianalyte detection of ß-agonists, a ß2-adrenergic receptor (ß2-AR) was cloned from Syrian hamster lung and heterogeneously expressed by Spodoptera frugiperda (Sf9) cells. The recombinant ß2-AR was purified from intracellular soluble proteins of infected Sf9 cells, and was utilized to establish an enzyme-linked-receptor assay (ELRA) to detect a group of ß-agonists simultaneously. This assay was based on direct competitive inhibition of binding of horseradish peroxidase-labeled ractopamine to the immobilized ß2-AR proteins by ß-agonists. The IC50 and limit of detection values for ractopamine were 30.38µgL(-1) and 5.20µgL(-1), respectively. Clenbuterol and salbutamol showed 87.7% and 58.5% cross-reactivities with ractopamine, respectively. This assay is simple, rapid, and environmentally friendly, showing a potential application in the screening of ß-agonists in animal feeds.
Assuntos
Agonistas Adrenérgicos beta/análise , Técnicas Biossensoriais/métodos , Enzimas de Restrição do DNA/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Baculoviridae/genética , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Mesocricetus , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMO
Antimicrobial peptides (AMPs), both synthetic and from natural sources, have raised interest recently as potential alternatives to antibiotics. Cyto-insectotoxin (Cit1a) is a 69-amino-acid antimicrobial peptide isolated from the venom of the central Asian spider Lachesana tarabaevi. The synthetic gene Cit1a fused with the enhanced green fluorescent protein (EGFP) gene was expressed as the EGFP-Cit1a fusion protein using a cysteine protease-deleted Bombyx mori nucleopolyhedrovirus (BmNPV-CP(-)) bacmid in silkworm larva and pupa. The antimicrobial effect of the purified protein was assayed using disk diffusion and broth microdilution methods. The minimum inhibitory concentration of EGFP-Cit1a was also measured against several bacterial strains and showed similar antimicrobial activity to that of the synthetic Cit1a reported earlier. The EGFP-Cit1a fusion protein showed antibiotic activity toward gram-positive and gram-negative bacteria at the micromolar concentration level. These results show that active Cit1a can be produced and purified in silkworm, although this peptide is insecticidal. This study demonstrates the potential of active Cit1a purified from silkworms to use as an antimicrobial agent.
Assuntos
Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/metabolismo , Aracnídeos/enzimologia , Bactérias/efeitos dos fármacos , Venenos de Aranha/química , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Aracnídeos/genética , Baculoviridae/genética , Bombyx/virologia , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Larva/virologia , Testes de Sensibilidade Microbiana , Pupa/virologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite.
Assuntos
Calmodulina/metabolismo , Leishmania/metabolismo , Movimento , Miosinas/química , Miosinas/metabolismo , Fosfolipídeos/metabolismo , Conformação Proteica , Área Sob a Curva , Baculoviridae , Dimerização , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Microscopia Eletrônica de Transmissão , Oligonucleotídeos/genética , PlasmídeosRESUMO
BACKGROUND: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. RESULTS: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. CONCLUSIONS: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.
Assuntos
Antígenos Virais/isolamento & purificação , Baculoviridae/genética , Biotecnologia/métodos , Proteínas do Capsídeo/isolamento & purificação , Flexiviridae/genética , Doenças das Plantas/virologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Alho/virologia , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Spodoptera/virologiaRESUMO
Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.
Assuntos
Difosfato de Adenosina/metabolismo , Amônia/metabolismo , Glutamato Desidrogenase/metabolismo , Baculoviridae/genética , Linhagem Celular , DNA Complementar/biossíntese , DNA Complementar/genética , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , NADP/metabolismoRESUMO
BACKGROUND: Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. METHODS: A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3'-5'-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman's assay in microplates. RESULTS: A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA. CONCLUSIONS: Results presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition.
Assuntos
Acetilcolinesterase/genética , Insetos Vetores/enzimologia , Leishmaniose/transmissão , Phlebotomus/enzimologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Inibidores da Colinesterase/farmacologia , DNA Complementar/química , DNA Complementar/genética , Feminino , Humanos , Insetos Vetores/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Cinética , Leishmaniose/parasitologia , Masculino , Dados de Sequência Molecular , Organofosfatos/farmacologia , Phlebotomus/genética , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , TexasRESUMO
High quantity and quality of recombinant Ca(2+)-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca(2+)-dependent cellular signaling pathways. S100A3 is a unique member of the S100 protein family with the highest cysteine content (10%). This protein, derived from human hair follicles and cuticles, is characterized by an N-terminal acetyl group and irreversible posttranslational citrullination by peptidylarginine deiminase causing its homotetramer assembly. Insect cells, capable of introducing eukaryotic N-terminus and disulfide bonds, are an appropriate host in which to express this cysteine-rich protein. Four out of ten cysteines in the recombinant S100A3 form two intramolecular disulfide bridges that modulate its Ca(2+)-affinity. Three free thiol groups located at the C-terminus are predicted to form the high-affinity Zn(2+)-binding site. Citrullination of specific arginine residues in native S100A3 can be mimicked by site-directed mutagenic substitution of Arg/Ala. This chapter details our procedures used for the purification and characterization of the human S100A3 protein and its pseudo citrullinated forms expressed in insect cells.
Assuntos
Citrulina/metabolismo , Cisteína , Processamento de Proteína Pós-Traducional , Proteínas S100/genética , Proteínas S100/isolamento & purificação , Animais , Baculoviridae/genética , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/genética , DNA Recombinante/genética , Dissulfetos/química , Expressão Gênica , Vetores Genéticos/genética , Recombinação Homóloga , Humanos , Mutagênese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas S100/química , Proteínas S100/metabolismo , Células Sf9 , SpodopteraRESUMO
Host protein synthesis is shut down in the lytic baculovirus expression vector system (BEVS). This also affects host proteins involved in routing secretory proteins through the endoplasmic reticulum (ER)-Golgi system. It has been demonstrated that a secretory alkaline phosphatase-EGFP fusion protein (SEFP) can act as a traceable and sensitive secretory reporter protein in BEVS. In this study, a chaperone, calreticulin (CALR), and the translation initiation factor eIF4E were co-expressed with SEFP using a bicistronic baculovirus expression vector. We observed that the intracellular distribution of SEFP in cells co-expressing CALR was different from co-expressing eIF4E. The increased green fluorescence emitted by cells co-expressing CALR had a good correlation with the abundance of intracellular SEFP protein and an unconventional ER expansion. Cells co-expressing eIF4E, on the other hand, showed an increase in extracellular SEAP activity compared to the control. Utilization of these baculovirus expression constructs containing either eIF4E or CALR offers a significant advantage for producing secreted proteins for various biotechnological and therapeutic applications.
Assuntos
Baculoviridae/genética , Calreticulina/genética , Fator de Iniciação 4E em Eucariotos/genética , Chaperonas Moleculares/genética , Fosfatase Alcalina/genética , Animais , Calreticulina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Genes Reporter , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Insetos/citologia , Insetos/genética , Insetos/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
Since 2003, the highly pathogenic avian influenza (HPAI) H5N1 has become a serious problem in animals and an increasing threat to public health. To develop effective vaccines for H5 HPAI in chickens, virus-like particles (VLP) were produced using a baculovirus expression system. The particles comprised hemagglutinin (HA) alone (HA-VLP) or HA in combination with a matrix protein (M1; HAM-VLP) derived from a recent clade 2.3.2.1 H5N1 HPAI virus. To compare the immunogenicity and protective efficacy of these VLPs, 10 µg HAM-VLP, the equivalent amounts of HA incorporated HA-VLP or whole inactivated virus (WIV), were emulsified with mineral oil and used to immunize chickens. The serum hemagglutination inhibition antibody levels induced by HA-VLP and HAM-VLP were comparable to WIV. Antibodies to nucleoprotein were detected only in the WIV group. Immunized chickens in each group survived and were protected against a lethal homologous virus challenge, showing no clinical signs of infection. The challenge virus was detected intermittently in some oropharyngeal swabs, but not in cloacal swabs or various organs, which means that VLPs and WIV provide protection against systemic but not local virus replication in chickens. After the challenge, the HA-VLP group showed significantly increased serum antibody levels compared to the HAM-VLP and WIV groups, and some chickens in the HA-VLP group seroconverted with respect to nucleoprotein. Taken together, these results suggest that VLPs may be an effective method for controlling HPAI in chickens. They could be applied to a differentiating infected from vaccinated animals (DIVA) strategy. In addition, it is likely that HAM-VLP is more efficacious than HA-VLP in chickens.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Baculoviridae/genética , Baculoviridae/imunologia , Galinhas , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Células Sf9 , Organismos Livres de Patógenos Específicos , Vacinação/veterinária , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas da Matriz Viral/genética , Vírion/genética , Vírion/imunologiaRESUMO
The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been widely used not only to achieve a high level of foreign gene expression in insect cells but also for efficient gene transduction into mammalian cells without any replication. In addition to the efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. The baculovirus has abundant CpG motifs in the viral genome and is capable of inducing pro-inflammatory cytokines and interferons (IFNs) through Toll-like receptor (TLR)-dependent and -independent signaling pathways in a cell-type-specific manner. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I) and MDA5 (melanoma-differentiation-associated gene 5) have been identified as viral dsRNA detectors and the adaptor IPS-1 (IFN-beta promoter stimulator-1) interacts with RIG-I and MDA5 to facilitate type-I IFN production mediated interferon regulatory factor 3 (IRF3) and 7 (IRF7). These helicases and IPS-1, however, were not essential for the type-I IFN and inflammatory cytokine responses to baculovirus. The baculovirus also has a strong adjuvant activity, and recombinant baculoviruses encoding neutralization epitopes elicit protective immunity in mice. This review deals with the current status of our knowledge of the induction of host innate immune responses by baculovirus and discusses the future prospects for baculovirus vectors.